Amine-derivatized nucleosides and oligonucleosides

ABSTRACT

Nucleotides and oligonucleosides functionalized to include alkylamino functionality, and derivatives thereof. In certain embodiments, the compounds of the invention further include steroids, reporter molecules, reporter enzymes, lipophilic molecules, peptides or proteins attached to the nucleotides through the alkylamino group.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.08/117,363, filed Sep. 3, 1993, now U.S. Pat. No. 6,783,931 which is acontinuation-in-part of application serial number PCT/US92/09196, filedOct. 23, 1992 (now application Ser. No. 08/211,882, filed Apr. 22,1994), which is a continuation-in-part, of application Ser. No.07/782,374, filed Oct. 24, 1991 now abandoned, which is acontinuation-in-part of application Ser. No. 07/463,358, filed Jan. 11,1990, now abandoned and of application Ser. No.07/566,977, filed Aug.13, 1990 now abandoned. The entire disclosures of each of theseapplications, which are assigned to the assignee of this application,are incorporated herein by reference.

FIELD OF THE INVENTION

This application is directed to nucleosides, oligonucleotides andoligonucleosides functionalized to include alkylamino functionality, andderivatives thereof. In certain embodiments, the compounds of theinvention further include steroids, reporter molecules, reporterenzymes, lipophilic molecules, peptides or proteins attached to thenucleosides through the alkylamino group.

BACKGROUND OF THE INVENTION

Messenger RNA (mRNA) directs protein synthesis. Antisense methodology isthe complementary hybridization of relatively short oligonucleotides tomRNA or DNA such that the normal, essential functions of theseintracellular nucleic acids are disrupted. Hybridization is thesequence-specific hydrogen bonding via Watson-Crick base pairs ofoligonucleotides to RNA or single-stranded DNA. Such base pairs are saidto be complementary to one another.

The naturally occurring events that provide the disruption of thenucleic acid function, discussed by Cohen in Oligonucleotides: AntisenseInhibitors of Gene Expression, CRC Press, Inc., Boca Raton, Fla. (1989)are thought to be of two types. The first, hybridization arrest, denotesthe terminating event in which the oligonucleotide inhibitor binds tothe target nucleic acid and thus prevents, by simple steric hindrance,the binding of essential proteins, most often ribosomes, to the nucleicacid. Methyl phosphonate oligonucleotides (Miller, et al., Anti-CancerDrug Design 1987, 2, 117) and α-anomer oligonucleotides are the two mostextensively studied antisense agents which are thought to disruptnucleic acid function by hybridization arrest.

The second type of terminating event for antisense oligonucleotidesinvolves the enzymatic cleavage of the targeted RNA by intracellularRNase H. A 2′-deoxyribofuranosyl oligonucleotide or oligonucleotideanalog hybridizes with the targeted RNA and this duplex activates theRNase H enzyme to cleave the RNA strand, thus destroying the normalfunction of the RNA. Phosphorothioate oligonucleotides are the mostprominent example of an antisense agent that operates by this type ofantisense terminating event.

Considerable research is being directed to the application ofoligonucleotides and oligonucleotide analogs as antisense agents fordiagnostics, research reagents and potential therapeutic purposes. Atleast for therapeutic purposes, the antisense oligonucleotides andoligonucleotide analogs must be transported across cell membranes ortaken up by cells to express activity. One method for increasingmembrane or cellular transport is by the attachment of a pendantlipophilic group.

Ramirez, et al., J. Am. Chem. Soc. 1982, 104, 5483, introduced thephospholipid group 5′-O-(1,2-di-O-myristoyl-sn-glycero-3-phosphoryl)into the dimer TpT independently at the 3′ and 5′ positions.Subsequently Shea, et al., Nuc. Acids Res. 1990, 18, 3777, disclosedoligonucleotides having a 1,2-di-O-hexyldecyl-rac-glycerol group linkedto a 5′-phosphate on the 5′-terminus of the oligonucleotide. Certain ofthe Shea, et. al. authors also disclosed these and other compounds inpatent application PCT/US90/01002. A further glucosyl phospholipid wasdisclosed by Guerra, et al., Tetrahedron Letters 1987, 28, 3581.

In other work, a cholesteryl group was attached to the inter-nucleotidelinkage between the first and second nucleotides (from the 3′ terminus)of an oligonucleotide. This work is disclosed in U.S. Pat. No. 4,958,013and further by Letsinger, et al., Proc. Natl. Acad. Sci. USA 1989, 86,6553. The aromatic intercalating agent anthraquinone was attached to the2′ position of a sugar fragment of an oligonucleotide as reported byYamana, et al., Bioconjugate Chem. 1990, 1, 319. The same researchersplaced pyrene-1-methyl at the 2′ position of a sugar (Yamana et. al.,Tetrahedron Lett. 1991, 32, 6347).

Lemairte, et al., Proc. Natl. Acad. Sci. USA 1986, 84, 648; andLeonetti, et al., Bioconjugate Chem. 1990, 1, 149. The 3′ terminus ofthe oligonucleotides each include a 3′-terminal ribose sugar moiety. Thepoly(L-lysine) was linked to the oligonucleotide via periodate oxidationof this terminal ribose followed by reduction and coupling through aN-morpholine ring. Oligonucleotide-poly(L-lysine) conjugates aredescribed in European Patent application 87109348.0. In this instancethe lysine residue was coupled to a 5′ or 3′ phosphate of the 5′ or 3′terminal nucleotide of the oligonucleotide. A disulfide linkage has alsobeen utilized at the 3′ terminus of an oligonucleotide to link a peptideto the oligonucleotide as is described by Corey, et al., Science 1987,238, 1401; Zuckermann, et al., J. Am. Chem. Soc. 1988, 110, 1614; andCorey, et al., J. Am. Chem. Soc. 1989, 111, 8524.

Nelson, et al., Nuc. Acids Res. 1989, 17, 7187 describe a linkingreagent for attaching biotin to the 3′-terminus of an oligonucleotide.This reagent, N-Fmoc-O-DMT-3-amino-1,2-propanediol is now commerciallyavailable from Clontech Laboratories (Palo Alto, Calif.) under the name3′-Amine on. It is also commercially available under the name3′-Amino-Modifier reagent from Glen Research Corporation (Sterling,Va.). This reagent was also utilized to link a peptide to anoligonucleotide as reported by Judy, et al., Tetrahedron Letters 1991,32, 879. A similar commercial reagent (actually a series of such linkershaving various lengths of polymethylene connectors) for linking to the5′-terminus of an oligonucleotide is 5′-Amino-Modifier C6. Thesereagents are available from Glen Research Corporation (Sterling, Va.).These compounds or similar ones were utilized by Krieg, et al.,Antisense Research and Development 1991, 1, 161 to link fluorescein tothe 5′-terminus of an oligonucleotide. Other compounds of interest havealso been linked to the 3′-terminus of an oligonucleotide. Asseline, etal., Proc. Natl. Acad. Sci. USA 1984, 81, 3297 described linkingacridine on the 3′-terminal phosphate group of an poly (Tp)oligonucleotide via a polymethylene linkage. Haralambidis, et al.,Tetrahedron Letters 1987, 28, 5199 report building a peptide on a solidstate support and then linking an oligonucleotide to that peptide viathe 3′ hydroxyl group of the 3′ terminal nucleotide of theoligonucleotide. Chollet, Nucleosides & Nucleotides 1990, 9, 957attached an Aminolink 2 (Applied Biosystems, Foster City, Calif.) to the5′ terminal phosphate of an oligonucleotide. They then used thebifunctional linking group SMPB (Pierce Chemical Co., Rockford, Il) tolink an interleukin protein to the oligonucleotide.

An EDTA iron complex has been linked to the 5 position of a pyrimidinenucleoside as reported by Dreyer, et al., Proc. Natl. Acad. Sci. USA1985, 82, 968. Fluorescein has been linked to an oligonucleotide in thesame manner as reported by Haralambidis, et al., Nucleic Acid Research1987, 15, 4857 and biotin in the same manner as described in PCTapplication PCT/US/02198. Fluorescein, biotin and pyrene were alsolinked in the same manner as reported by Telser, et al., J. Am. ChemSoc. 1989, 111, 6966. A commercial reagent, Amino-Modifier-dt, from GlenResearch Corporation (Sterling, Va.) can be utilized to introducepyrimidine nucleotides bearing similar linking groups intooligonucleotides.

Cholic acid linked to EDTA for use in radioscintigraphic imaging studieswas reported by Betebenner, et.al., Bioconjugate Chem. 1991, 2, 117;however, it is not known to link cholic acid to nucleosides, nucleotidesor oligonucleotides.

OBJECTS OF THE INVENTION

It is one object of this invention to provide nucleosides,oligonucleotides and oligonucleosides that include alkylamino chemicalfunctionality.

It is a further object of the invention to provide compounds havingimproved transfer across cellular membranes.

It is another object to provide compounds that include intercalators,nucleic acid cleaving agents, cell surface phospholipids, and/ordiagnostic agents.

It is yet another object to provide improvements in research anddiagnostic methods and materials for assaying bodily states in animals,e specially disease states.

It is an additional object of this invention to provide therapeutic andresearch materials having improved transfer and uptake properties forthe treatment of diseases through modulation of the activity of DNA orRNA.

BRIEF DESCRIPTION OF THE INVENTION

These and other objects are satisfied by the present invention, whichprovides compounds containing alkylamino chemical functionality. In oneaspect, the invention provides nucleosides having base portions andribofuranosyl sugar portions. Such nucleosides bear at a 2O-position, a3′-position, or a 5′-O-position a substituent having formula:—R_(A)—N(R_(1a)) (R_(1b))where:

-   -   R_(A) is alkyl having from 1 to about 10 carbon atoms or R_(A)        is (CH₂—CH₂—Q—)_(x);    -   R_(1a) and R_(1b), independently, are H, R_(A), R₂, or an amine        protecting group or have formula C(X)—R₂, C(X)—R_(A)—R₂,        C(X)—Q—R_(A)-R₂, C(X)—Q—R₂;    -   R₂ includes a steroid molecule, a reporter molecule, a        lipophilic molecule, a reporter enzyme, a peptide, a protein, or        has formula —Q—(CH₂CH₂—Q—)_(x)—R₃;    -   X is O or S;    -   each Q is, independently, is NH, O, or S;    -   x is 1 to about 200;    -   R₃ is H, R_(A), C(O)OH, C(O)OR_(A), C(O)R₄, R_(A)—N₃, R_(A)—NH₂,        or R_(A)—SH; and    -   R₄ is Cl, Br, I, SO₂R₅ or has structure:    -   m is 2 to 7; and    -   R₅ is alkyl having 1 to about 10 carbon atoms.

In another aspect, the invention provides oligonucleotides andoligonucleosides comprising a plurality of linked nucleosides, whereineach nucleoside includes a ribofuranosyl sugar portion and a baseportion and at least one (preferably more than one) of the nucleosidesbears at a 2′-O-position, a 3′-O-position, or a 5′-O-position asubstituent having formula —R_(A)—N(R_(1a)) (R_(1b)).

In another aspect the invention provides methods for preparingoligonucleotides and oligonucleosides comprising the steps of contactingnucleosides according to the invention for a time and under reactionconditions effective to form a covalent bond therebetween. In preferredembodiments, at least one of the nucleosides bears a phosphoramidategroup at its 2′-O-position or at its 3′-O-position.

In other embodiments, compounds according to the invention are preparedby contacting a nucleoside, oligonucleotide or oligonucleoside withderivatizing reagents. For example, a nucleoside, oligonucleotide oroligonucleoside bearing a 2′-hydroxy group, a 3′-hydroxy group, or a5′-hydroxy group under basic conditions with a compound having formulaL₁—R_(A)—N(R_(1a)) (R_(1b)) wherein L₁ is a leaving group such as ahalogen and at least one of R_(1a) and R_(1b) is an amine protectinggroup.

The present invention also provides methods for inhibiting theexpression of particular genes in the cells of an organism, comprisingadministering to said organism a compound according to the invention.Also provided are methods for inhibiting transcription and/orreplication of particular genes or for inducing degradation ofparticular regions of double stranded DNA in cells of an organism byadministering to said organism a compound of the invention. Furtherprovided are methods for killing cells or virus by contacting said cellsor virus with a compound of the invention. The compound can be includedin a composition that further includes an inert carrier for thecompound.

BRIEF DESCRIPTION OF THE FIGURES

The numerous objects and advantages of the present invention may bebetter understood by those skilled in the art by reference to theaccompanying figures, in which:

FIG. 1 is a graph showing mouse plasma concentrations of a controlcompound and two of the compounds of the invention. The plasmaconcentration is plotted verses time.

FIG. 2 is a three dimensional graph showing distribution of a controlcompound among various tissue in the mouse. Specific tissues are shownon one axis, time on a second axis and percent of dose on the thirdaxis. The compound was delivered by intravenous injections.

FIG. 3 is a three dimensional graph showing distribution of a compoundof the invention among various tissue in the mouse. Specific tissues areshown on one axis, time on a second axis and percent of dose on thethird axis. The compound was delivered by intravenous injection.

FIG. 4 is a three dimensional graph showing distribution of a furthercompound of the invention among various tissue in the mouse. Specifictissues are shown on one axis, time on a second axis and percent of doseon the third axis. The compound was delivered by intravenous injection.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides nucleosides, oligonucleotides andoligonucleosides containing alkylamino chemical functionality. Thenucleoside subunits can be “natural” or “synthetic” moieties. Eachnucleoside is formed from a naturally occurring or synthetic base and anaturally occurring or synthetic pentofuranosyl sugar group.

The term “oligonucleotide” refers to a polynucleotide formed from aplurality of linked nucleotide units. The nucleotides units each includea nucleoside unit. In the context of this invention, the term“oligonucleoside” refers to a plurality of nucleoside units that arelinked together. In a generic sense, since each nucleotide unit of anoligonucleotide includes a nucleoside therein, the term“oligonucleoside” can be considered to be inclusive of oligonucleotides(i.e., nucleosides linked together via phosphate linking groups). In afurther sense, the term “oligonucleoside” also refers to a plurality ofnucleosides that are linked together via linkages other than phosphatelinkages. The term “oligonucleoside” thus effectively includes naturallyoccurring species or synthetic species formed from naturally occurringsubunits. For brevity, the term “oligonucleoside” will be used asencompassing both phosphate linked (oligonucleotides) and non-phosphatelinked polynucleoside species.

Oligonucleosides according to the invention also can include modifiedsubunits. Representative modifications include modification of aheterocyclic base portion of a nucleoside or a sugar portion of anucleoside. Exemplary modifications are disclosed in the followingUnited States Patent Applications: Ser. No. 463,358, filed Jan. 11,1990, entitled Compositions And Methods For Detecting And Modulating RNAActivity; Ser. No. 566,977, filed Aug. 13, 1990, entitled Sugar ModifiedOligonucleotides That Detect And Modulate Gene Expression; Ser. No.558,663, filed Jul. 27, 1990, entitled Novel Polyamine ConjugatedOligonucleotides; Ser. No. 558,806, filed Jul. 27, 1991, entitledNuclease Resistant Pyrimidine Modified Oligonucleotides That Detect AndModulate Gene Expression and Ser. No. PCT/US91/00243, filed Jan. 11,1991, entitled Compositions and Methods For Detecting And Modulating RNAActivity. Each of these patent applications are assigned to the assigneeof this invention. The disclosure of each is incorporated herein byreference.

The term oligonucleoside thus refers to structures that include modifiedportions, be they modified sugar moieties or modified base moieties,that function similarly to natural bases and natural sugars.Representative modified bases include deaza or aza purines andpyrimidines used in place of natural purine and pyrimidine bases;pyrimidines having substituent groups at the 5 or 6 position; andpurines having altered or replacement substituent groups at the 2, 6 or8 positions. Representative modified sugars include carbocyclic oracyclic sugars, sugars having substituent groups at their 2′ position,and sugars having substituents in place of one or more hydrogen atoms ofthe sugar. Other altered base moieties and altered sugar moieties aredisclosed in U.S. Pat. No. 3,687,808 and PCT application PCT/US89/02323.

Altered base moieties or altered sugar moieties also include othermodifications consistent with the spirit of this invention. Sucholigonucleosides are best described as being structurallydistinguishable from yet functionally interchangeable with naturallyoccurring or synthetic wild type oligonucleotides. All sucholigonucleosides are comprehended by this invention so long as theyfunction effectively to mimic the structure of a desired RNA or DNAstrand.

For use in antisense methodology, the oligonucleosides of the inventionpreferably comprise from about 10 to about 30 subunits. It is morepreferred that such oligonucleosides comprise from about 15 to about 25subunits. As will be appreciated, a subunit is a base and sugarcombination suitably bound to adjacent subunits through, for example, aphosphorous-containing (e.g., phosphodiester) linkage or some otherlinking moiety. The nucleosides need not be linked in any particularmanner, so long as they are covalently bound. Exemplary linkages arethose between the 3′- and 5′-positions or 2′- and 5′-positions ofadjacent nucleosides. Exemplary linking moieties are disclosed in thefollowing references: Beaucage, et al., Tetrahedron 1992, 48, 2223 andreferences cited therein; and U.S. patent applications: Ser. No.703,619, filed May 21, 1991; Ser. No. 903,160, filed Jun. 24, 1992; Ser.No. 039,979, filed Mar. 20, 1993; Ser. No. 039,846, filed Mar. 30, 1993;and Ser. No. 040,933, filed Mar. 31, 1993. Each of the foregoing patentapplications are assigned to the assignee of this invention. Thedisclosure of each is incorporated herein by reference.

It is preferred that the RNA or DNA portion which is to be modulatedusing oligonucleosides of the invention be preselected to comprise thatportion of DNA or RNA which codes for the protein whose formation oractivity is to be modulated. The targeting portion of the composition tobe employed is, thus, selected to be complementary to the preselectedportion of DNA or RNA, that is, to be an antisense oligonucleoside forthat portion.

In accordance with one preferred embodiment of this invention, thecompounds of the invention hybridize to HIV mRNA encoding the tatprotein, or to the TAR region of HIV mRNA. In another preferredembodiment, the compounds mimic the secondary structure of the TARregion of HIV mRNA, and by doing so bind the tat protein. Otherpreferred compounds are complementary to sequences for herpes, papillomaand other viruses.

The nucleosides and oligonucleosides of the invention can be used indiagnostics, therapeutics and as research reagents and kits. They can beused in pharmaceutical compositions by including a suitablepharmaceutically acceptable diluent or carrier. They further can be usedfor treating organisms having a disease characterized by the undesiredproduction of a protein. The organism should be contacted with anoligonucleotide having a sequence that is capable of specificallyhybridizing with a strand of nucleic acid coding for the undesirableprotein. Treatments of this type can be practiced on a variety oforganisms ranging from unicellular prokaryotic and eukaryotic organismsto multicellular eukaryotic organisms. Any organism that utilizesDNA-RNA transcription or RNA-protein translation as a fundamental partof its hereditary, metabolic or cellular control is susceptible totherapeutic and/or prophylactic treatment in accordance with theinvention. Seemingly diverse organisms such as bacteria, yeast,protozoa, algae, all plants and all higher animal forms, includingwarm-blooded animals, can be treated. Further, since each cell ofmulticellular eukaryotes can be treated since they include both DNA-RNAtranscription and RNA-protein translation as integral parts of theircellular activity. Many of the organelles (e.g., mitochondria andchloroplasts) of eukaryotic cells also include transcription andtranslation mechanisms. Thus, single cells, cellular populations ororganelles can also be included within the definition of organisms thatcan be treated with therapeutic or diagnostic oligonucleotides. As usedherein, therapeutics is meant to include the eradication of a diseasestate, by killing an organism or by control of erratic or harmfulcellular growth or expression.

In one aspect, the present invention is directed to nucleosides andoligonucleosides that bear at least one amine-containing substituent ata position. Such substituents preferably have formula —R_(A)—N(R_(1a))(R_(1b)) and are appended at 2′-O—, 3′—O—, and/or 5′—O— positions.

Each R_(A) according to the invention is an alkyl moiety independentlyselected to having 1 to about 10 carbon atoms or R_(A) is a polyether, apolythioether or polyalkylamine. The term “alkyl” is intended to includestraight chain and branched hydrocarbons. The preferred length of thesehydrocarbons is 1 to about 7 carbon atoms.

R_(1a) and R_(1b) according to the invention are H, R_(A), R₂, an amineprotecting group, or have formula C(X)—R₂, C(X)—R_(A)—R₂,C(X)—Q—R_(A)—R₂, C(X)—Q—R₂. Protecting groups are known per se aschemical functional groups that can be selectively appended to andremoved from functionalities, such as amine groups. These groups arepresent in a chemical compound to render such functionality inert tochemical reaction conditions to which the compound is exposed. See,e.g., Greene and Wuts, Protective Groups in Organic Synthesis, 2dedition, John Wiley & Sons, New York, 1991. Numerous amine protectinggroups are known in the art, including, but not limited to: phthalimide(PHTH), trifluoroacetate (triflate), allyloxycarbonyl (Alloc),benzyloxycarbonyl (CBz), chlorobenzyloxycarbonyl, t-butyloxycarbonyl(Boc), fluorenylmethoxycarbonyl (Fmoc), and isonicotinyloxycarbonyl(i-Noc) groups. (see, e.g., Veber and Hirschmann, et al., J. Org. Chem.1977, 42, 3286 and Atherton, et al., The Peptides, Gross and Meienhofer,Eds, Academic Press; New York, 1983; Vol. 9 pp. 1-38).

R₂ can include a steroid molecule, a reporter molecule, a lipophilicmolecule, a reporter enzyme, a peptide, a protein (i.e., a substituentconsisting essentially of same), or a molecule having formula—Q—(CH₂CH₂—Q—)_(x)—R₃. For the purposes of this invention the terms“reporter molecule” and “reporter enzyme” are inclusive of thosemolecules or enzymes that have physical or chemical properties thatallow them to be identified in gels, fluids, whole cellular systems,broken cellular systems and the like utilizing physical properties suchas spectroscopy, radioactivity, colorimetric assays, fluorescence, andspecific binding. Steroids, include those chemical compounds thatcontain a perhydro-1,2-cyclopentanophenanthrene ring system. Proteinsand peptides are utilized in their usual sense as polymers of aminoacids. Normally peptides comprise such polymers that contain a smallernumber of amino acids per unit molecule than do the proteins. Lipophilicmolecules include naturally-occurring and synthetic aromatic andnon-aromatic moieties such as fatty acids, esters, alcohols and otherlipid molecules, substituted aromatic groups such as dinitrophenylgroups, cage structures such as adamantane and buckminsterfullerenes,and aromatic hydrocarbons such as benzene, perylene, phenanthrene,anthracene, naphthalene, pyrene, chrysene, and naphthacene.

Particularly useful as steroid molecules are the bile acids includingcholic acid, deoxycholic acid and dehydrocholic acid; steroids includingcortisone, digoxigenin, testosterone and cholesterol and even cationicsteroids such as cortisone having a trimethylaminomethyl hydrazide groupattached via a double bond at the 3 position of the cortisone rings.Particularly useful as reporter molecules are biotin, dinitrophenyl, andfluorescein dyes. Particularly useful as lipophilic molecules arealicyclic hydrocarbons, saturated and unsaturated fatty acids, waxes,terpenes and polyalicyclic hydrocarbons including adamantane andbuckminsterfullerenes. Particularly useful as reporter enzymes arealkaline phosphatase and horseradish peroxidase. Particularly useful aspeptides and proteins are sequence-specific peptides and proteinsincluding phosphodiesterase, peroxidase, phosphatase and nucleaseproteins. Such peptides and proteins include SV40 peptide, RNaseA, RNaseH and Staphylococcal nuclease. Particularly useful as terpenoids arevitamin A, retinoic acid, retinal and dehydroretinol.

R₂ also can have formula —Q—(CH₂CH₂—Q—)_(x)—R₃, where Q is O, S, or NH.Subscript x can be 1 to about 200, preferably about 20 to about 150,more preferably about 10 to about 50. Preferably, Q are selected to beO, such that R₂ constitutes a poly(ethyleneglycol) (PEG) group (i.e.,R₃═H) or a functionalized derivative thereof (e.g., R₃═C(O)Cl). R₃ canbe H, R_(A), C(O)OH, C(O)OR_(A), C(O)R₄, R_(A)—N₃, R_(A)—NH₂ or R_(A)—SHwhere R₄ is F, Cl, Br, I, SO₂R₅ or a small thio-containing heterocyclehaving structure:

where m is 2 to 7. Representative PEG-containing R₂ groups are disclosedby Ouchi, et al., Drug Design and Discovery 1992, 9, 93, Ravasio, etal., J. Org. Chem. 1991, 56, 4329, and Delgardo et. al., CriticalReviews in Therapeutic Drug Carrier Systems 1992, 9, 249.

Oligonucleosides according to the invention can be assembled in solutionor through solid-phase reactions, for example, on a suitable DNAsynthesizer utilizing nucleosides according to the invention and/orstandard nucleotide precursors. The nucleosides and nucleotideprecursors can already bear alkylamino groups or can be later modifiedto bear such groups.

In the former case, compounds according to the invention are preparedby, for example, reacting nucleosides bearing at least one free 2′-,3′-, or 5′-hydroxyl group under basic conditions with a compound havingformula L₁—(CH₂)_(n)—N(R_(1a)) (R_(1b)) where L₁ is a leaving group andat least one of R_(1a) and R_(1b) is an amine protecting group.Displacement of the leaving group through nucleophilic attack of anoxygen anion produces the desired amine derivative. Leaving groupsaccording to the invention include but are not limited to halogen,alkyl-sulfonyl, substituted alkylsulfonyl, arylsulfonyl, substitutedarylsulfonyl, hetercyclcosulfonyl or trichloroacetimidate. A morepreferred group includes chloro, fluoro, bromo, iodo,p-(2,4-dinitroanilino)benzenesulfonyl, benzenesulfonyl, methyl-sulfonyl(mesylate)., p-methylbenzenesulfonyl (tosylate), p-bromobenzenesulfonyl,trifluoromethylsulfonyl (triflate), trichloroacetimidate, acyloxy,2,2,2-trifluoroethanesulfonyl, imidazolesulfonyl, and2,4,6-trichlorophenyl, with bromo being preferred.

Suitably protected nucleosides can be assembled into an oligonucleosidesaccording to known techniques. See, e.g., Beaucage, et al., Tetrahedron1992, 48, 2223.

Oligonucleosides according to the invention also can be prepared byassembling an oligonucleoside and appending alkyl-amino functionalitythereto. For example, oligonucleosides having free hydroxyl groups canbe assembled according to known techniques and then reacted with areagent having formula L₁—(CH₂)_(n)—N(R_(1a)) (R_(1b)). As will berecognized, however, greater selectivity can be achieved in terms ofplacement of alkylamino functionality within an oligonucleoside byintroducing such functionality, as discussed above, on selectednucleosides and then using both the selected nucleosides and othernucleosides to construct an oligonucleoside.

Once assembled, an oligonucleoside bearing one or more groups havingformula —R_(A)—N(R_(1a)) (R_(1b)) wherein at least one of R_(1a) andR_(1b) is a protecting group is treated with reagents effective toremove the protecting group. Once deprotected, the oligonucleoside canbe contacted with electrophillic moieties such as, for example,succinimidyl esters and other activated carboxylic acids includingC(═O)—O-succinimide and C(═O)—O-pentafluorophenyl, isothiocyanates,sulfonyl chlorides, halacetamides, phospholipid carbocyclic acid activeesters, o-phenanthroline-5-iodoacetamide, fluorescein isothiocyanate,1-pyrene butyric acid-N-hydroxy succinimide ester and carboxylic acidderivatives of PNA (carboxylic acid derivatives of peptide nucleicacids). Preferred electrophillic moieties includecholesteryl-3-hemisuccinate-N-hydroxy succinimide ester,pyrene-1-butyric acid-N-hydroxy succinimide ester and polyethyleneglycol-propionic acid-N-hydroxy succimide ester.

Thus, the invention first builds the desired linked nucleoside sequencein the normal manner on the DNA synthesizer. One or more (preferably twoor more) of the linked nucleosides are then functionalized orderivatized with the lipophilic steroid, reporter molecule, lipophilicmolecule, reporter enzyme, peptide or protein.

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing examples, which are not intended to be limiting. Alloligonucleotide sequences are listed in a standard 5′ to 3′ order fromleft to right.

EXAMPLE 1 Oligonucleotides Having 2′-Protected-Amine Terminating LinkingGroup

A. Preparation of5′-Dimethoxytrityl-2′-(O-Pentyl-N-phthalimido)-2′-DeoxyadenosinePhosphoramidite.

To introduce a functionalization at the 2′ position of nucleotideswithin desired oligonucleotide sequences,5′-dimethoxytrityl-2′-(O-pentyl-N-phthalimido)-2′-deoxyadenosinephosphoramidite was utilized to provide a linking group attached to the2′ position of nucleotide components of an oligonucleotide. Thiscompound was synthesized generally in accordance with the procedures ofpatent application serial numbers US91/00243 and 463,358, identifiedabove, starting from adenosine. Briefly, this procedure treats adenosinewith NaH in dimethylformamide (DMF) followed by treatment withN-(5-bromopentyl)phthalimide. Further treatment with (CH₃)₃SiCl,Ph—C(O)—Cl and NH₄OH yields N6-benzyl protected 2′-pentyl-N-phthalimidofunctionalized adenosine. Treatment with DIPA and CH₂Cl₂ adds a DMTblocking group at the 5′ position. Finally phosphitylation gives thedesired phosphoramidite compound. This compound was utilized in the DNAsynthesizer as a 0.09M solution in anhydrous CH₃CN. Oligonucleotidesynthesis was carried out in either an ABI 390B or an ABI 394synthesizer employing the standard synthesis cycles with an extendedcoupling time of 10 minutes during coupling of Compound 2 into theoligonucleotide sequence. Coupling efficiency of greater than 98% wasobserved.

B. 2′-Protected-Amine Linking Group Containing PhosphodiesterOligonucleotides

The following oligonucleotides having phosphodiester inter-nucleotidelinkages were synthesized:

-   -   Oligomer 9: 5′ TA*G 3′;    -   Oligomer 10: 5′ CCA* G 3′;    -   Oligomer 11 (SEQ ID NO:1): 5′ GGC TGA* CTG CG 3′;    -   Oligomer 12 (SEQ ID NO:2): CTG TCT CCA* TCC TCT TCA CT;    -   Oligomer 13: CTG TCT CCA* TCC TCT TCA* CT        wherein A* represents a nucleotide functionalized to incorporate        a pentyl-N-phthalimido functionality. Oligomers 12 and 13 are        antisense compounds to the E2 region of the bovine papilloma        virus-1 (BPV-1). Oligomers 12 and 13 have the same sequence as        Oligomer 3 in Application Serial Number 782,374, except for the        2′ modification. The oligonucleotides were synthesized in either        a 10 μmol scale or a 3×1 μmol scale in the “Trityl-On” mode.        Standard deprotection conditions (30% NH₄OH, 55° C., 24 hours)        were employed. The oligonucleotides were purified by reverse        phase HPLC (Waters Delta-Pak C₄15 μm, 300A, 25×100 mm column        equipped with a guard column of the same material). They were        detritylated and further purified by size exclusion using a        Sephadex G-25 column. NMR analyses by both proton and phosphorus        NMR confirmed the expected structure for the oligomers 9 and 10.

C. 2′-Protected-Amine Linking Group Containing PhosphorothioateOligonucleotides

The following oligonucleotides having phosphorothioate inter-nucleotidelinkages were synthesized:

-   -   Oligomer 14 (SEQ ID NO:3):    -   T_(s)T_(s)G_(s) C_(s)T_(s)T_(s) C_(s)C_(s)A*_(s) T_(s)C_(s)T_(s)        T_(s)C_(s)C_(s) T_(s)C_(s)G_(s) T_(s)C;    -   Oligomer 15 (SEQ ID NO:4):    -   T_(s)G_(s)G_(s) G_(s)A_(s)G_(s)C_(s)C_(s)A_(s) T_(s)A_(s)G_(s)        C_(s)G_(s)A*_(s) G_(s)G_(s)C; and    -   Oligomer 16:    -   T_(s)G_(s)G_(s) G_(s)A*_(s)G_(s) C_(s)C_(s)A*_(s)        T_(s)A*_(s)G_(s) C_(s)G_(s)A*_(s) G_(s)G_(s)C        wherein A* represents a nucleotide functionalized to incorporate        a pentyl-N-phthalimido functionality and the subscript “s”        represents a phosphorothioate inter-nucleotide backbone linkage.        Oligomer 14 is an antisense compound directed to the E2 region        of the bovine papilloma virus-1 (BPV-1). Oligomers 15 and 16 are        antisense compounds to ICAM. Oligomer 14 has the same sequence        as Oligomer 3 in Application Serial Number 782,374, except for        the 2′ modification whereas Oligomers 15 and 16 have the same        sequence as Oligomer 4 in Application Serial Number 782,374        except for the 2′ modification. These oligonucleotides were        synthesized as per the method of Example 1(B) except during the        synthesis, for oxidation of the phosphite moieties, the Beaucage        reagent (i.e., 3H-1,2-benzodithioate-3-one 1,1-dioxide, see,        Radhakrishnan, et al., J. Am. Chem. Soc. 1990, 112, 1253) was        used as a 0.24 M solution in anhydrous CH₃CN solvent. The        oligonucleotides were synthesized in the “Trityl-On” mode and        purified by reverse phase HPLC utilizing the purification        procedure of Example 1 (B).

D. 2′-O-Methyl Derivatized, 2′-Protected-Amine Linking Group ContainingRNA Oligonucleotides

The following oligonucleotides having 2′-O-methyl groups on eachnucleotide not functionalized with a 2′-protected aminefunctionalization were synthesized:

-   -   Oligomer 17: CCA A*GC CUC AGA; and (SEQ ID NO:24)    -   Oligomer 18: CCA GGC UCA GA*T (SEQ ID NO:25)        wherein A* represents a nucleotide functionalized to incorporate        a pentyl-N-phthalimido functionality and where the remaining        nucleotides except the 3′-terminus nucleotide are each        2′-O-methyl derivatized nucleotides. The 3′-terminus nucleotide        in both Oligomers 17 and 18 is a 2′-deoxy nucleotide. Both        oligomers 17 and 18 are antisense compounds to the HIV-1 TAR        region. The oligonucleotides were synthesized as per the method        of Example 6 in Application Serial Number 782,374 (utilizing        Compound 2 thereof) for introduction of the nucleotides        containing the pentyl-N-phthalimido functionality and        appropriate 2-O-methyl phosphoramidite nucleotides from        Chemgenes Inc. (Needham, Mass.) for the remaining RNA        nucleotides. The 3′-terminus terminal 2′-deoxy nucleotides were        standard phosphoamidites utilized for the DNA synthesizer. The        oligonucleotides were deprotected and purified as per the method        of Example 1(B).

EXAMPLE 2 Functionalization of Oligonucleotides at the 2′ Position

A. Functionalization with Biotin

1. Single Site Modification

About 10 O.D. units (A₂₆₀) of Oligomer 12 (see, Example 1)(approximately 60 nmols based on the calculated extinction coefficientof 1.6756×10⁵) was dried in a microfuge tube. The oligonucleotide wasdissolved in 200 μl of 0.2 M NaHCO₃ buffer andD-biotin-N-hydroxysuccinimide ester (2.5 mg, 7.3 μmols) (Sigma, St.Louis, Mo.) was added followed by 40 μl DMF. The solution was let standovernight. The solution was applied to a Sephadex G-25 column (0.7×15cm) and the oligonucleotide fractions were combined. Analytical HPLCshowed nearly 85% conversion to the product. The product was purified byHPLC (Waters 600E with 991 detector, Hamilton PRP-1 column 0.7×15 cm;solvent A: 50 mM TEAA pH 7.0; B: 45 mM TEAA with 80% acetonitrile: 1.5ml flow rate: Gradient: 5% B for first 5 minutes, linear (1%) increasein B every minute thereafter) and further desalted on Sephadex G-25 togive the oligonucleotide:

Oligomer 19:CTG TCT CCA* TCC TCT TCA CT wherein A* represents anucleotide functionalized to incorporate a biotin functionality linkedvia a 2′-O-pentyl-amino linking group to the 2′ position of thedesignated nucleotide. HPLC retention times are shown in Table 1 below.

2. Multiple Site Modification

About 10 O.D. units (A₂₆₀) of Oligomer 13 (see, Example 1, approximately60 nmols) was treated utilizing the method of Example 8(A) (1) inApplication Serial Number 782,374 with D-biotin-N-hydroxysuccinimideester (5 mg) in 300 μl of 0.2 M NaHCO₃ buffer/50 Ml DMF. Analytical HPLCshowed 65t of double labeled oligonucleotide product and 30% of singlelabeled products (from the two available reactive sites). HPLC andSephadex G-25 purification gave the oligonucleotide:

Oligomer 20: CTG TCT CCA TCC TCT TCA* CT wherein A* representsnucleotides functionalized to incorporate a biotin functionality linkedvia a 2′-O-pentyl-amino linking group to the 2′ position of thedesignated nucleotide. HPLC retention times for this product (and itsaccompanying singly labeled products) are shown in Table 1 below.

B. Functionalization with Fluorescein

1. Single Site Modification

A 1M Na₂CO₃/1M NaHCO₃ buffer (pH 9.0) was prepared by adding 1M NaHCO₃to 1 M Na₂CO₃. A 200 μl portion of this buffer was added to 10 O.D.units of oligomer 12 (see, Example 1) in a microfuge tube. A 10 mgportion of fluorescein-isocyanate in 500 μl DMF was added to give a 0.05M solution. A 100 μl portion of the fluorescein solution was added tothe oligonucleotide solution in the microfuge tube. The tube was coveredwith aluminum foil and let stand overnight. The reaction mixture wasapplied to a Sephadex G-25 column (0.7×20 cm) that had been equilibratedwith 25% (v/v) ethyl alcohol in water. The column was eluted with thesame solvent. Product migration could be seen as a yellow band wellseparated from dark yellow band of the excess fluorescein reagent. Thefractions showing absorption at 260 nm and 485 nm were combined andpurified by HPLC as per the purification procedure of Example 2 (A) (1).Analytical HPLC indicated 81% of the desired doubly functionalizedoligonucleotide. The product was lyophilized and desalted on Sephadex togive the oligonucleotide:

Oligomer 21Q (SEQ ID NO:2): CTG TCT CCA* TCC TCT TCA CT wherein A*represents a nucleotide functionalized to incorporate a fluoresceinfunctionality linked via a 2′-O-pentyl-amino linking group to the 2′position of the designated nucleotide. HPLC retention times are shown inTable 1 below.

2. Multiple Site Modification

A 10 O.D. unit (A₂₆₀) portion of oligomer 13 (from Example 1) wasdissolved in 300 μl of the 1M Na₂HCO₃/1M Na₂CO₂ buffer of Example 2(B)(1) and 200 μl of the fluorescein-isothiocyanate stock solution ofExample 2(B) (1) was added. The resulting solution was treated as perExample 2(B)(1). Analytical HPLC indicated 61% of doubly labeled productand 38% of singly labeled products. Work up of the reaction gave theoligonucleotide:

Oligomer 22: CTG TCT CCA* TCC TCT TCA* CT wherein A* representsnucleotides functionalized to incorporate a fluorescein functionalitylinked via a 2′-O-pentyl-amino linking group to the 2′ position of thedesignated nucleotide. HPLC retention times are shown in Table 1 below.

C. Functionalization with Cholic Acid

1. Single Site Modification

A 10 O.D. unit (A₂₆₀) portion of oligomer 12 (see, Example 1) wastreated with cholic acid-NHS ester (Compound 1 in Application SerialNumber 782,374, 5 mg, 9.9 μmols) in 200 μl of 0.2 M NaHCO₃ buffer/40 μlDMF. The reaction mixture was heated for 16 hours at 45° C. The productwas isolated as per the method of Example 2 (B) (1). Analytical HPLCindicated greater than 85% product formation. Work up of the reactiongave the oligonucleotide:

Oligomer 23: CTG TCT CCA* TCC TCT TCA CT wherein A* represents anucleotide functionalized to incorporate a cholic acid functionalitylinked via a 2′-O-pentyl-amino linking group to the 2′ position of thedesignated nucleotide. HPLC retention times are shown in Table 1 below.

2. Multiple Site Modification

A 10 O.D. unit (A₂₆₀) portion of Oligomer 13 (see, Example 1) wastreated with cholic acid-NHS ester (Compound 1 in Application SerialNumber 782,374, 10 mg, 19.8 μmols) in 300 μl of 0.2 M NaHCO₃ buffer/50μl DMF. The reaction mixture was heated for 16 hours at 45° C. Theproduct was isolated as per the method of Example 2(A)(1). AnalyticalHPLC revealed 58% doubly labeled product, 17% of a first singly labeledproduct and 24% of a second singly labeled product. Work up as perExample 2(A)(1) gave the oligonucleotide:

-   -   Oligomer 24: CTG TCT CCA* TCC TCT TCA* CT        wherein A* represents nucleotides functionalized to incorporate        a cholic acid functionality linked via a 2′-O-pentyl-amino        linking group to the 2′ position of the designated nucleotide.        HPLC retention times are shown in Table 1 below.

D. Functionalization with Digoxigenin

1. Single Site Modification

A 10 O.D. unit (A₂₆₀) portion of Oligomer 12 (see, Example 1) wastreated with digoxigenin-3-O-methylcarbonyl-E-aminocaproic N-hydroxysuccinimide ester (Boehringer Mannheim Corporation, Indianapolis, Ind.)in 200 μl of 0.1 M borate pH 8.3 buffer/40 μl DMF. The reaction mixturewas let stand overnight. The product was isolated as per the method ofExample 2(A) (1). Work up of the reaction gave the oligonucleotide:

-   -   Oligomer 25: CTG TCT CCA* TCC TCT TCA CT        wherein A* represents a nucleotide function alized to        incorporate a digoxigenin functionality linked via a        2′-O-pentyl-amino linking group to the 2′ position of the        designated nucleotide. HPLC retention times are shown in Table 1        below.

2. Multiple Site Modification

A 10 O.D. units (A₂₆₀) portion of Oligomer 13 (see, Example 1) wastreated with digoxigenin-3-O-methylcarbonyl-ε-aminocaproic N-hydroxysuccinimide ester (Boehringer Mannheim Corporation, Indianapolis, Ind.)in 300 μl of 0.1 M borate pH 8.3 buffer/50 μl DMF. The reaction mixturewas let stand overnight. The product was isolated as per the method ofExample 2 (A) (1). Work up as per Example 2 (A) (1) gave theoligonucleotide:

-   -   Oligomer 26: CTG TCT CCA* TCC TCT TCA* CT        wherein A* represents nucleotides functionalized to incorporate        a cholic acid functionality linked via a 2′-O-pentyl-amino        linking group to the 2′ position of the designated nucleotide.        HPLC retention times are shown in Table 1 below.

TABLE 1 HPLC Retention Times Of Oligonucleotides Functionalized At 2′Position Retention Time Minutes Oligomer Mono Substitution MultipleSubstitution Oligomer 12¹ 21.78 Oligomer 13¹ 22.50 Oligomer 19² 23.58Oligomer 20² 24.16^(a) 25.19^(b) Oligomer 21³ 26.65 Oligomer 22³26.99^(a) 29.33b 27.55^(a) Oligomer 23⁴ 30.10 Oligomer 24⁴ 30.38^(a)37.00^(b) 32.22^(a) Oligomer 25⁵ 28.06 Oligomer 26⁵ 28.14^(a) 33.32^(b)29.24^(a) Conditions: Waters 600E with 991 detector, Hamilton PRP-1column 0.7 × 15 cm; solvent A: 50 mM TEAA pH 7.0; B: 45 mM TEAA with 80%acetonitrile: 1.5 ml flow rate: Gradient: 5% B for first 5 minutes,linear (1%) increase in B every minute thereafter; ^(a)Mono conjugatedminor product; ^(b)Doubly conjugated major product; ¹ParentOligonucleotide - no 2′ functionalization; ²2′ Biotin functionalization;³2′ Fluorescein functionalization; ⁴2′ Cholic Acid functionalization;and ⁵2′ Digoxigenin functionalization.Procedure AConfirmation of Structure of Functionalized Oligonucleotides Containinga Tethered 2′-Amino Moiety

Oligonucleotides of the invention were digested with snake venomphosphodiesterase and calf-intestine alkaline phosphatase to theirindividual nucleosides. After digestion, the nucleoside composition wasanalyzed by HPLC. The HPLC analysis established that functionalizednucleotide compounds having the tethered 2′-amino moiety thereon werecorrectly incorporated into the oligonucleotide.

Snake venom phosphodiesterase [Boehringer-Mannheim cat. #108260, 1 mg(1.5 units)/0.5 ml] and alkaline phosphatase from calf intestine (1unit/microliter, Boehringer-Mannheim cat. # 713023) in Tris-HCl buffer(pH 7.2, 50 mM) were used to digest the oligonucleotides to theircomponent nucleosides. To 0.5 O.D. units of oligonucleotide in 50 μlbuffer (nearly 40 μM final concentration for a 20 mer) was added 5 μl ofsnake venom phosphodiesterase (nearly 0.3 units/mL, final concentration)and 10 μl of alkaline phosphatase (app. 150 units/mL, finalconcentration). The reaction mixture was incubated at 37° C. for 3hours. Following incubation, the reaction mixture was analyzed by HPLCusing a reverse phase analytical column (app. 30×2.5 cm); solvent A: 50mM TEAA pH 7; solvent B: acetonitrile; gradient 100% for 10 minutes,then 5% B for 15 minutes, then 10% B and then wash. The results of thesedigestion are shown in Table 2 for representative oligonucleotides.

TABLE 2 OLIGONUCLEOTIDE ANALYSIS VIA ENZYMATIC DIGESTION ObservedRatios** Abs. max. 267 252 267 260 Oligomer C G T A* A Oligomer 10 2 1 1Oligomer 11 3 5 2 1 Oligomer 12 9 1 8 1 1 Oligomer 13 9 1 8 2*Nucleoside having 2′-O-linker attached thereto; and **Corrected towhole numbers.

As is evident from Table 2, the correct nucleoside ratios are observedfor the component nucleotides of the test oligonucleotides.

Procedure B

Determination of Melting Temperatures (Tm's) of Cholic AcidOligonucleotide Conjugates

The relative ability of oligonucleotides to bind to their complementarystrand is compared by determining the melting temperature of thehybridization complex of the oligonucleotide and its complementarystrand. The melting temperature (Tm), a characteristic physical propertyof double helices, denotes the temperature in degrees centigrade atwhich 50% helical versus coil (un-hybridized) forms are present. Tm ismeasured by using the UV spectrum to determine the formation andbreakdown (melting) of hybridization. Base stacking, which occurs duringhybridization, is accompanied by a reduction in UV absorption(hypochromicity). Consequently a reduction in UV absorption indicates ahigher T_(m). The higher the Tm, the greater the strength of the bindingof the strands. Non-Watson-Crick base pairing has a strong destabilizingeffect on the Tm. Consequently, absolute fidelity of base pairing isnecessary to have optimal binding of an antisense oligonucleotide to itstargeted RNA.

1. Terminal End Conjugates

a. Synthesis

A series of oligonucleotides were synthesized utilizing standardsynthetic procedures (for un-functionalized oligonucleotides) or theprocedure of Example 3(A) in Application Serial Number 782,374 foroligonucleotides having a 5′-terminus amino linker bearingoligonucleotide or the procedure of Example 3(B) in Application SerialNumber 782,374 for 5′-terminus cholic acid-bearing oligonucleotides.Each of the oligonucleotides had the following 5-LO antisense sequence:5′ TCC AGG TGT CCG CAT C₃′(SEQ ID NO:6). The nucleotides weresynthesized on a 1.0 μmol scale. Oligomer 32 was the parent compoundhaving normal phosphodiester inter-nucleotide linkages. Oligomer 33incorporated phosphorothioate inter-nucleotide linkages in the basicoligonucleotide sequence. Oligomer 34 is a an intermediateoligonucleotide having a 5′-aminolink at the 5′-terminus of the basicoligonucleotide sequence and Oligomer 35 was a similar 5′-aminolinkcompound incorporating phosphorothioate inter-nucleotide linkages.Oligomer 36 is a 5′-terminus cholic acid conjugate of the basicphosphodiester oligonucleotide sequence while Oligomer 37 is a similar5′-cholic acid conjugate incorporating phosphorothioate inter-nucleotidelinkages. Oligomers 32 and 33 were synthesized in a “Trityl-On” mode andwere purified by HPLC. Oligomers 34 and 35 were synthesized as perExample 3ø(A) in Application Serial Number 782,374 without or withBeaucage reagent treatment, to yield phosphodiester or phosphorothioateinter-nucleotide linkages, respectively. Oligomers 36 and 37 wereprepared from samples of Oligomers 34 and 35, respectively, utilizing asolution of cholic acid N-hydroxysuccinimide ester (Compound 1) 1dissolved in DMF as per Example 3(B) in Application Serial Number782,374. Oligomers 36 and 37 were purified by HPLC. The products wereconcentrated and desalted in a Sephadex G-25 column. Gel electrophoresisanalyses also confirmed a pure product with the pure conjugate movingslower than the parent oligonucleotide or 5′-amino functionalizedoligonucleotide.

b. Melting Analysis

The test oligonucleotides [either the phosphodiester, phosphorothioate,cholic acid conjugated phosphodiester, cholic acid conjugatedphosphorothioate or 5′-aminolink intermediate phosphodiester orphosphorothioate oligonucleotides of the invention or otherwise] andeither the complementary DNA or RNA oligonucleotides were incubated at astandard concentration of 4 μM for each oligonucleotide in buffer (100mM NaCl, 10 mM Na-phosphate, pH 7.0, 0.1 mM EDTA). Samples were heatedto 90 degrees C. and the initial absorbance taken using a GuilfordResponse II spectrophotometer (Corning). Samples were then slowly cooledto 15 degrees C. and then the change in absorbance at 260 nm wasmonitored during the heat denaturation procedure. The temperature waselevated 1 degree/absorbance reading and the denaturation profileanalyzed by taking the 1st derivative of the melting curve. Data wasalso analyzed using a two-state linear regression analysis to determinethe Tm's. The results of these tests are shown in Table 3 as are theHPLC retention times of certain of the test compounds.

TABLE 3 Melting Temperature Of The Hybridization Complex Of TheOligonucleotide And Its Complementary Strand Tm** HPLC Ret. Time*Oligomer DNA RNA minutes 32 62.6 62.0 — 33 55.4 54.9 — 34 ND ND 13.6 35ND ND 17.0 36 63.4 62.4 22.0 37 56.3 55.8 22.5 *HPLC conditions: WaltersDelta Pak C-18 RP 2.5u column; at 0 min 100% 0.1 TEAA; at 30 min 50%TEAA and 50% Acetonitrile: Flow rate 1.0 ml/min. **Tm at 4 μM eachstrand from fit of duplicate melting curves to 2-state model with linearsloping base line. Conditions: 100 mM NaCl, 10 mM Phosphate, 0.1 mMEDTA, pH 7.0. ND = not determined

As is evident from Table 3, conjugates of cholic acid at the end of theoligonucleotide do not affect the Tm of the oligonucleotides.

2. Strands Incorporating 2′-O-Pentylamino Linker

a. Synthesis

An oligonucleotide of the sequence:

-   -   Oligomer 38 (SEQ ID NO:7): GGA* CCG GA*A* GGT A*CG A*G        wherein A* represents a nucleotide functionalized to incorporate        a pentylamino. functionality at its 2′-position was synthesized        in a one micromole scale utilizing the method of Example 1(B).        The oligonucleotide was purified by reverse phase HPLC,        detritylated and desalted on Sephadex G-25. PAGE gel analysis        showed a single band. A further oligonucleotide, Oligomer 39,        having the same sequence but without any 2′-O-amino linker was        synthesis in a standard manner. A complementary DNA        oligonucleotide of the sequence:

oligomer 40 (SEQ ID NO:8): CCT GGC CTT CCA TGC TC was also synthesizedin a standard manner as was a complementary RNA oligonucleotide of thesequence:

-   -   oligomer 41 (SEQ ID NO:9): CCU GGC CUU CCA UGC UC.

b. Melting Analysis

Melting analysis was conducted as per the method of Procedure B(1)(b).The results are shown in Table 4.

TABLE 4 Melting Temperature Of The Hybridization Complex Of TheOligonucleotide And Its Complementary Strand Tm* Oligomer DNA¹ RNA² 3854.5 58.0 39 60.6 56.9 *Tm at 4 μM each strand from fit of duplicatemelting curves to 2-state model with linear sloping base line.Conditions: 100 mM NaCl, 10 mM Phosphate, 0.1 mM EDTA, pH 7.0. ¹AgainstDNA complementary strand, Oligomer 40. ²Against RNA complementarystrand, Oligomer 41

As is evident from Table 4 against the RNA 40 complementary strand thechange in Tm's between the strand having 2′-amino linkers thereon andthe unmodified strand is 1.1 degrees (0.22 change per modification).Against the DNA strand, the change is −6.1 degrees (−1.2 change permodification). When compared to the parent unmodified oligonucleotidethe 2′-amino linker- containing strand has a stabilizing effect uponhybridization with RNA and a destabilizing effect upon hybridizationwith DNA.

Compounds of the invention were tested for their ability to increasecellular uptake. This was determined by judging either their ability toinhibit the expression of bovine papilloma virus-1 (BPV-1) or an assayinvolving luciferase production (for HIV-1).

Procedure C

Determination of Cellular Uptake Judged by the Inhibition of Expressionof Bovine Papilloma Virus-1 (bpv-1) as Measured by an E2 TransactivationAssay

For this test, a phosphorothioate oligonucleotide analog of thesequence:

-   -   Oligomer 42: CTG TCT CCA TCC TCT TCA CT        was used as the basic sequence. This sequence is designed to be        complementary to the translation initiation region of the E2        gene of bovine papilloma virus type 1 (BPV-1). Oligomer 42        served as the positive control and standard for the assay.        Oligomer 3 (from Example 4 in Application Serial Number 782,374)        served as a second test compound. It has the same basic sequence        except it is a phosphorothioate oligonucleotide and further it        has a cholic acid moiety conjugated at the 3′end of the        oligonucleotide. Oligomer 2 (from Example 2 in Application        Serial Number 782,374) served as a third test compound. Again it        is of the same sequence, it is a phosphorothioate        oligonucleotide and it has a cholic acid moiety conjugated at        the 5′-end. Oligomer 5 (from Example 5 in Application Serial        Number 782,374) served as a fourth test compound. Once again it        has the same sequence, is a phosphorothioate oligonucleotide and        it has a cholic acid moiety conjugated at both the 3′-end and        5′-end. A fifth test compound was a phosphorothioate        oligonucleotide with no significant sequence homology with        BPV-1. A sixth test compound was a further phosphorothioate        oligonucleotide with no significant sequence homology with        BPV-1. The last test compound, the seventh test compound, was a        phosphorothioate oligonucleotide with cholic acid conjugated to        the 3′-end but having no significant sequence homology with        BPV-1. Compounds five, six and seven served as negative controls        for the assay.

For each test I-38 cells were plated at 5×10⁴ cells per cm² in 60 mmpetri dishes. Eight hours after plating, medium was aspirated andreplaced with medium containing the test oligonucleotide and incubatedovernight. Following incubation, medium was aspirated and replaced withfresh medium without oligonucleotide and incubated for one hour. Cellswere then transfected by the CaPO₄ method with 2 μg of pE2RE-1-CAT.After a four hour incubation period cells were glycerol shocked (15%glycerol) for 1 minute followed by washing 2 times with PBS. Medium wasreplaced with DMEM containing oligonucleotide at the originalconcentration. Cells were incubated for 48 hours and harvested. Celllysates were analyzed for chloramphenicol acetyl transferase by standardprocedures. Acetylated and nonacetylated ¹⁴C-chloramphenicol wereseparated by thin layer chromatography and quantitated by liquidscintillation. The results are expressed as percent acetylation.

Two lots of the positive control compound were found to acetylate at alevel of 29% and 30%. The negative controls, test compounds five, sixand seven, were found to acetylate at 59%, 58% and 47%, respectively.The 3′-cholic acid conjugate test compound, oligomer 3, was found toacetylate to 23%, the 5′-cholic acid conjugate test compound, Oligomer2, was found to acetylate to 36% and the test compound conjugated atboth the 3′-end and the 5′-end, Oligomer 5, was found to acetylate to27%.

The results of this test suggests that placement of a cholic acid moietyat the 3′-terminus of an oligonucleotide increase the activity. This inturn suggests that the increased activity was the result of increasedcellular membrane transport.

Procedure D

Determination of Cellular Uptake Judged By Inhibition of pHIVluc WithCholic Acid Linked 2′-O-Methyl Substituted Oligonucleotides

For this test the absence of an oligonucleotide in a test well served asthe control. All oligonucleotides were tested as 2′-O-methyl analogs.For this test an oligonucleotide of the sequence:

Oligomer 43. (SEQ ID NO:10): CCC AGG CUC AGA where each of thenucleotides of the oligonucleotide includes a 2′-O-methyl substituentgroup served as the basic test compound. The second test compound of thesequence:

-   -   Oligomer 44: 5′-CHA CCC AGG CUC AGA        wherein CHA represents cholic acid and where each of the        nucleotides of the oligonucleotide includes a 2′-O-methyl        substituent group, was also of the same sequence as the first        test compound. This second test compound included cholic acid        conjugated to its 5′-end and was prepared as per the method of        Example 3 in Application Serial Number 782,374 utilizing        2′-O-methyl phosphoramidite intermediates as identified in        Example 1(C). The third test compound of the sequence:    -   Oligomer 45: CCC AGG CUC AGA 3′-CHA        wherein CHA represents cholic acid and where each of the        nucleotides of the oligonucleotide includes a 2′-O-methyl        substituent group was also of the same sequence as the first        test compound. The third test compound included cholic acid        conjugated to its 3′-end and was prepared as per the method of        Example 4 in Application Serial Number 782,374 utilizing        2′-O-methyl phosphoramidite intermediates as identified in        Example 1(C). The fourth test compound was a 2′-O-Me        oligonucleotide of a second sequence:    -   Oligomer 46 (SEQ ID NO:11): GAG CUC CCA GGC        where each of the nucleotides of the oligonucleotide includes a        2′-O-methyl substituent group. The fifth test compound was of        sequence:    -   Oligomer 47: 5′-CHA GAG CUC CCA GGC        wherein CHA represents cholic acid and where each of the        nucleotides of the oligonucleotide includes a 2′-O-methyl        substituent group. It was of the same sequence as the fifth test        compound. This test compound included cholic acid conjugated to        its 5′-end and was prepared as per the method of Example 3 in        Application Serial Number 782,374 utilizing 2′-O-methyl        phosphoramidite intermediates as identified in Example 1(C).

A sixth test compound was a randomized oligonucleotide of the sequence:

-   -   Oligomer 48 (SEQ ID NO:12): CAU GCU GCA GCC.

HeLa cells were seeded at 4×10⁵ cells per well in 6-well culture dishes.Test oligonucleotides were added to triplicate wells at 1 μM and allowedto incubate at 37° C. for 20 hours. Medium and oligonucleotide were thenremoved, cells washed with PBS and the cells were CaPO₄ transfected.Briefly, 5 μg of pHIVluc, a plasmid expressing the luciferase cDNA underthe transcriptional control of the HIV LTR constructed by ligating theKpnI/HindIII restriction fragments of the plasmids pT3/T71uc and pHIVpap(NAR 19(12)) containing the luciferase cDNA and the HIV LTRrespectively, and 6 μg of pcDEBtat, a plasmid expressing the HIV tatprotein under the control of the SV40 promoter, were added to 500 μl of250 mM CaCl₂, then 500 μl of 2×HBS was added followed by vortexing.After 30 minutes, the CaPO₄ precipitate was divided evenly between thesix wells of the plate, which was then incubated for 4 hours. The mediaand precipitate were then removed, the cells washed with PBS, and fresholigonucleotide and media were added. Incubation was continuedovernight. Luciferase activity was determined for each well thefollowing morning. Media was removed, then the cells washed 2× with PBS.The cells were then lysed on the plate with 200 μl of LB (1t Trit X-100,25 mM Glycylglycine pH 7.8, 15 mM MgSO₄, 4 mM EGTA, 1 mM DTT). A 75 μlaliquot from each well was then added to a well of a 96 well plate alongwith 75 μl of assay buffer (25 mM Glycylglycine pH 7.8, 15 mM MgSO₄, 4mM EGTA, 15 mM KPO₄, 1 mM DTT, 2.5 mM ATP). The plate was then read in aDynatec multiwell luminometer that injected 75 μl of Luciferin buffer(25 mM Glycylglycine pH 7.8, 15 mM MgSO₄, 4 mM EGTA, 4 mM DTT, 1 mMluciferin) into each well, immediately reading the light emitted (lightunits).

The random sequence compound (Oligomer 48) and the other non-cholicacid-conjugated test compounds (Oligomers 43 and 46) had comparableactivity. The 5′-conjugate of the first sequence (Oligomer 44) also hadactivity comparable to the non-conjugated compounds. The 5′-conjugate ofthe second sequence (Oligomer 47) showed a three-fold increase inactivity compared to the non-conjugated compounds and the 3′-conjugateof the first sequence (Oligomer 45) showed a further 3-fold increase inactivity compared to Oligomer 47.

All the test cholic acid-bearing oligonucleotides showed significantinhibition of luciferase production compared to non-cholic acid- bearingoligonucleotides. This suggests that the increased activity was theresult of increased cellular membrane transport of the cholicacid-bearing test oligonucleotides.

EXAMPLE 3 Preparation of5′-O-[Dimethoxytrityl]-2′-O-[hexyl-(Ω-N-phthalimido)amino]uridine and5′-O-[dimethoxytrityl]-3′-O-[hexyl(Ω-N-phthalimidoamino)uridine.

2′,3′-O-Dibutyl stannylene-uridine was synthesized according to theprocedure of Wagner et. al., J. Org. Chem., 1974, 39, 24. This compoundwas dried over P₂O₅ under vacuum for 12 hours. To a solution of thiscompound (29 g, 42.1 mmols) in 200 ml of anhydrous DMF were added (16.8g, 55 mmols) of 6-bromohexyl phthalimide and 4.5 g of sodium iodide andthe mixture was heated at 130° C. for 16 hours under argon. The reactionmixture was evaporated, co-evaporated once with toluene and the gummytar residue was applied on a silica column (500 g). The column waswashed with 2L of ethyl acetate (EtOAc) followed by eluting with 10%methanol (MeOH):90% EtOAc. The product, 2′- and 3′-isomers ofO-hexyl-Ω-N-phthalimido uridine, eluted as an inseparable mixture(R_(f)=0.64 in 10% MeOH in EtOAc). By ¹³C NMR, the isomeric ration wasabout 55% of the 2′ isomer and about 45% of the 3′ isomer. The combinedyield was 9.2 g (46.2%). This mixture was dried under vacuum andre-evaporated twice with pyridine. It was dissolved in 150 mL anhydrouspyridine and treated with 7.5 g of dimethyocytrityl chloride (22.13mmols) and 500 mg of dimethylaminopyridine (DMAP). After 2 hour, thinlayer chromatography (TLC; 6:4 EtOAc:Hexane) indicated completedisappearance of the starting material and a good separation between 2′and 3′ isomers (R_(f)=0.29 for the 2′ isomer and 0.12 for the 3′isomer). The reaction mixture was quenched by the addition of 5 mL ofCH₃OH and evaporated under reduced pressure. The residue was dissolvedin 300 mL CH₂Cl₂, washed successively with saturated NaHCO₃ followed bysaturated NaCl solution. It was dried over Mg₂SO₄ and evaporated to give15 g of a brown foam which was purified on a silica gel (500 g) to give6.5 g of the 2′-isomer and 3.5 g of the 3′ isomer.

EXAMPLE 4 Preparation of5′-O-Dimethoxytrityl-2′-O-[hexyl-(Ω-N-phthalimido)amino]uridine-3′-O-(2-cyanoethyl-N,N-diisopropyl)-phosphoramidite.

The 5′-dimethoxytrityl-2′-[O-hexyl-(Ω-N-phthalimido)-amino]uridine (4 g,5.2 mmole) was dissolved in 40 mL of anhydrous CH₂Cl₂. To this solutiondiisopropylaminetetra-zolide (0.5 g, 2.9 mmol) was added and stirredovernight. TLC (1:1 EtoAC/hexane) showed complete disappearance ofstarting material. The reaction mixture was transferred with CH₂Cl₂ andwashed with saturated NaHCO₃ (100 mL) followed by saturated NaClsolution. The organic layer was dried over anhydrous Na₂SO₄ andevaporated to yield 6.4 g of a crude product which was purified in asilica column (200 g) using 1:1 hexane/EtOAc to give 4.6 g (4.7 mmol,90%) of the desired phosphoramidite.

EXAMPLE 5 Preparation of5′-O-(Dimethoxytrityl)-3′-O-[hexyl-(Ω-N-phthalimido) amino]uridine-2′-O-succinyl-aminopropyl controlled pore glass.

Succinylated and capped aminopropyl controlled pore glass (CPG; 500 Åpore diameter, aminopropyl CPG, 1.0 grams prepared according to Damhaet. al., Nucl. Acids Res. 1990, 18, 3813.) was added to 12 ml anhydrouspyridine in a 100 ml round-bottom flask.1-(3-Dimethylaminopropyl)-3-ethyl-carbo-diimide (DEC; 0.38 grams, 2.0mmol)], triethylamine (TEA; 100 μl , distilled over CaH₂),dimethylaminopyridine (DMAP; 0.012 grams, 0.1 mmol) and nucleoside5′-O-dimethoxytrityl-3′-O-[hexyl-(Ω-N-phthalimidoamino)]uridine (0.6grams, 0.77 mmol) were added under argon and the mixture shakenmechanically for 2 hours. More nucleoside (0.20 grams) was added and themixture shaken an additional 24 hours. CPG was filtered off and washedsuccessively with dichloromethane, triethylamine, and dichloromethane.The CPG was then dried under vacuum, suspended in 10 ml piperidine andshaken 15 minutes. The CPG was filtered off, washed thoroughly withdichloromethane and again dried under vacuum. The extent of loading(determined by spectrophotometric assay of dimethoxytrityl cation in 0.3M p-toluenesulfonic acid at 498 nm) was approximately 28 μmol/g. The5′-O-(dimethoxytrityl)-3′-O-[hexyl-(Ω-N-phthal-imidoamino]uridine-2′-O-succinyl-aminopropylcontrolled pore glass was used to synthesize the oligomers 5′-GACU*-3′and 5′-GCC TTT CGC GAC CCA ACA CU*-3′ (SEQ ID NO:13) (where the *indicated the derivatized nucleotide) in an ABI 380B DNA synthesizerusing phosphoramidite chemistry standard conditions. 45 and 200 O.D.'sof the 4-mer and 20-mer, respectively, were obtained from two and three1 μmol syntheses after purification by RP-HPLC and desalting.

The oligomer 5-GACU*-3′ was used to confirm the structure of3′-O-hexylamine tether introduced into the oligonucleotide by NMR. Asexpected a multiplet signal was observed between 1.0-1.8 ppm in ¹H NMR.The oligomer 5-GCC TTT CGC GAC CCA ACA CU* -3′ belongs to a HCV sequenceand it was used to show the nuclease resistance properties of the3′-O-amino tether [see example 38].

EXAMPLE 6 Preparation of5′-O-(Dimethoxytrityl)-2′-O-[hexyl-(Ω-N-phthalimido) amino]3′—O-succinyl-aminopropyl Controlled Pore Glass

The procedure of Example 5 was repeated, except that5′-O-(Dimethoxytrityl)-2′-O-[hexyl-(Ω-N-phthalimidoamido)-amino]uridinewas used in the loading process.

EXAMPLE 7 Preparation of5′-O-(Dimethoxytrityl)-2′-O-(hexylamino)-uridine

5′-O-(dimethoxytrityl)-2′-O-[hexyl-(Ω-N-phthalimido amino)]uridine (4.5grams, 5.8 mmol) was dissolved in 200 ml methanol in a 500 ml flask.Hydrazine (1 ml, 31 mmol) was added to the stirring reaction mixture.The mixture was heated to 60-65° in an oil bath and refluxed 14 hours.Solvent was evaporated in vacuo. The residue was dissolved indichloromethane (250 ml) and extracted twice with an equal volume NH₄OH.The organic layer was evaporated to yield 4.36 grams of crude product,and NMR indicated that the product was not completely pure. R_(f)=0 in100% ethyl acetate. The product was used in subsequent reactions withoutfurther purification.

EXAMPLE 8 Preparation of 5′-O-(dimethoxytrityl)-3′-O-[hexylamino]Uridine.

The procedure of Example 7 was repeated, except that5′-O-(dimethoxytrityl)-3′-O-[hexyl-(Ω-N-phthalimido-amino)] uridine wasused as the starting material.

EXAMPLE 9 Preparation of 5′-O-(dimethoxytrityl)-2′-O-[hexyl-N-(1-pyrenepropyl carbonyl)Amino]Uridine

5′-O-Dimethoxytrityl-2′-O-(hexylamino)uridine (0.5g, 0.78 mmol) wasdissolved in anhydrous DMF (15 mL). 1-Hydroxybenzotriazole (0.16 grams,1.17 mmol) and 1-pyrene-butyric acid pentafluorophenyl ester (0.53grams, 1.17 mmol) were added to the reaction mixture. The mixture wasstirred under argon at room temperature for 2 hours, after which it wasconcentrated in vacuo. Residual DMF was coevaporated with toluene. Theresidue was dissolved in dichloromethane (50 mL) and washed with anequal volume saturated NaHCO₃. The aqueous layer was washed withdichloromethane and the combined organic extracts washed with an equalvolume saturated NaCl. The aqueous layer was washed with dichloromethaneand the combined organic layers dried over MgSO₄ and concentrated. Theresidue was chromatographed on a silica gel column, eluting with agradient of 50% ethyl acetate in hexanes to 100% ethyl acetate. Thedesired product (0.83 grams, 58t) eluted with 100% ethyl acetate (R_(f)0.46 by thin-layer chromatography (TLC)).

EXAMPLE 10 Preparation of 5′-O-[Dimethoxytrityl]-2′-O-[hexyl-N-(1-pyrenepropylCarbonyl)amino]uridine-3′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite

5′-O-[Dimethoxytrityl]-2′-O-[hexyl-N-(1-pyrene propyl carbonyl)amino]uridine (0.80 grams, 0.87 mmol) was dissolved in 20 mL drydichloromethane. 2-CyanoethylN,N,N′,N′-tetra-isopropylphosphorodiamidite (purchased from SigmaChemical Co; 800 μL, 2.4 mmol) and diisopropylamine tetrazolide (0.090grams, 0.52 mmol) were added to the mixture, which was stirred underargon for 20 hours The reaction mixture was then concentrated in vacuoand the residue dissolved in dichloromethane (75 mL). The solution waswashed with an equal volume of saturated NaHCO₃. The aqueous layer waswashed with dichloromethane (20 mL) and the combined organic layerswashed with an equal volume of saturated NaCl. The aqueous layer waswashed with dichloromethane (20 mL) and the combined organic layersdried over MgSO₄ and concentrated. The residue was chromatographed on asilica gel column, eluting with a gradient of 50k ethyl acetate inhexanes to 100% ethyl acetate. The desired product (750 mg, 78% yield,R_(f) 0.54 by TLC in 100% ethyl acetate) eluted with 100% ethyl acetate.

EXAMPLE 11 Preparation of 2′-O-[hexyl-N-(1-pyrene-propyl-carbonyl)amino] Uridine.

5′—O-dimethoxytrityl-2′-O-[hexyl-N-(1-pyrene-propyl-carbonyl)amino]uridine(1.0 g) was dissolved in 20 mL CH₂Cl₂ and kept in ice-bath for 10minutes. To the cold solution, 5 mL of 80% acetic acid in water wasadded and the solution was left to stand for 30 minutes. It was thenevaporated to dryness and loaded into a silica column and eluted with10% methanol in methylene chloride to give2′-O-[hexyl-N-(1-pyrene-propyl-carbonyl)amino]uridine.

EXAMPLE 12 Preparation of 5′—O-(dimethoxytrityl)-2′—O-[hexyl-N-(1-pyrenepropylcarbonyl) amino] uridine-3′-O-[succinylaminopropyl]-controlledPore Glass

Succinylated/capped aminopropyl controlled pore glass was dried undervacuum for 3 hours immediately before use. A portion (0.3 g) was addedto 3 ml anhydrous pyridine in a 50 ml round-bottom flask. DEC (0.12grams, 0.63 mmol), TEA (25 μl, distilled over CaH₂), DMAP (0.005 grams,mmol) and 5′-O-(dimethoxytrityl)-3′-O-[hexyl-N-(1-pyrene propylcarbonyl]amino]uridine (0.21 grams, 0.22 mmol) were added under argonand the mixture shaken mechanically for 19 hours. More nucleoside (0.025grams) was added and the mixture shaken an additional 5.5 hours.Pentachlorophenol (0.045 grams, mmol) was added and the mixture shaken18 hours. CPG was filtered off and washed successively withdichloromethane, triethylamine, and dichloromethane. The resulting CPGwas then dried under vacuum, suspended in 15 ml piperidine and shaken 30minutes. CPG was filtered off, washed thoroughly with dichloromethaneand again dried under vacuum. The extent of loading (determined byspectrophotometric assay of dimethoxytrityl cation in 0.3 Mp-toluenesulfonic acid at 498 nm) was approximately 27 μmol/g. Theproduct solid support was subsequently used to synthesize the oligomers.

EXAMPLE 13 Preparation of 5′-O-dimethoxytrityl-3′-O-[hexyl-N-(1-pyrenepropyl carbonyl]amino] uridine-2′-O-(succinyl amino propyl) ControlledPore Glass

The procedure of Example 12 is repeated, except that5′-O-dimethoxytrityl-3′-O-[hexyl-N-(1-pyrenepropylcarbonyl]amino]uridine is used.

EXAMPLE 14 Preparation of 5′-O-(dimethoxytrityl)-2′-O-[hexyl-N-(5-thiocarbonyl-3,6-dipivolyl-fluorescein)amino]Uridine

Fluorescein isothiocyanate (Isomer I, available from Cal Biochem, LaJolla, Calif.) was treated with 12 equivalents of pivolyl chloride inEt₃N/THF to give di-O-pivolyl fluorescein isothiocyanate. This compoundwas purified in silica gel column using 3:1 hexane:ethyl acetate.Nucleoside 5′-O-(dimethoxytrityl)-2′-O-(hexylamino)uridine was thencondensed with dipivolyl fluorescein isothiocyanate inCH₂Cl₂/pyrimidine. The resultant compound5′-O-(dimethoxytrityl)-2′-O-[hexyl-N-(5-thiocarbonyl-3,6-dipivolyl-fluorescein)amino] uridine is then purified by using 100% ethyl acetate, in a silicacolumn.

EXAMPLE 15 Preparation of5′-O-dimethoxytrityl-2′-O-[hexyl-N-(5-thiocarbonyl-3,6-di-pivolylfluorescein) amino] Uridine-3′-O-(2-cyanoethyl, N—N-diisopropylPhosphoramidite

5′-O-(dimethoxytrityl)-2′-O-[hexyl-N-(5-thiocarbonyl-3,6-dipivolylfluorescein)amino]uridine (0.75 grams, 0.672 mmol) was dissolved in drydichloromethane (20 mL). 2-CyanoethylN,N,N′,N′-tetraisopropylphosphorodiamidite (700 μL, 2.2 mmol) anddiisopropylamine tetrazolide were added to the mixture, which wasstirred under argon for 16 hours. The reaction mixture was thenconcentrated in vacuo and the residue dissolved in dichloromethane (75mL) followed by washing with an equal volume of saturated NaHCO₃. Theaqueous layer was washed with dichloromethane (50 mL) and the combinedorganic layers washed with an equal volume of saturated NaCl. Theaqueous layer was washed with dichloromethane (50 mL) and the combinedorganic layers dried over MgSO₄ and concentrated. The residue waschromatographed on a silica gel column, eluting with a gradient of 25%ethyl acetate in hexanes to 100% ethyl acetate. The desired product (670mg, 77% yield, R_(f) 0.79 by TLC) eluted with 100% ethyl acetate.

EXAMPLE 16 Preparation of5′-O-(dimethoxytrityl)-2′-O-[hexyl-N-(5-thiocarbonyl-3,6-di-pivolylfluorescein)amino]uridine-3′—O-(succinylaminopropyl) controlled poreglass.

Succinylated and capped aminopropyl controlled pore glass (CPG) is driedunder vacuum for 3 hours immediately before use. CPG (0.3 grams) isadded to 3 ml anhydrous pyridine in a 50 ml round-bottom flask. DEC(0.12 grams, 0.63 mmol), TEA (25 μl, distilled over CaH₂, DMAP (dimethylamino pyridine) (0.005 grams, 0.04 mmol) and5′-O-dimethoxytrityl-2′-O-[hexyl-N-(5-thiocarbonyl-3,6-di-pivolylfluorescein) amino] uridine (0.21 grams, 0.19 mmol) are added underargon and the mixture shaken mechanically for 19 hours. More nucleoside(0.025 grams) is added and the mixture shaken an additional 5.5 hours.Pentachlorophenol (0.045 grams, 0.17 mmol) is added and the mixtureshaken 18 hours. CPG is filtered off and washed successively withdichloromethane, triethylamine, and dichloromethane. CPG then is driedunder vacuum, suspended in 15 mL piperidine and shaken 30 minutes. CPGis filtered off, washed thoroughly with dichloromethane, and again driedunder vacuum. The extent of loading is then determined byspectrophotometric assay of dimethoxytrityl cation in 0.3 Mp-toluenesulfonic acid at 498 nm.

EXAMPLE 17 Preparation of5′-O-(dimethoxytrityl)-2′-O-[hexyl-N-(3-oxycarbonyl-cholesteryl)amino]Uridine

Nucleoside 5′-O-(dimethoxytrityl)-2′-O-[hexylamino]-uridine (3.85 g, 6.0mmol) was dissolved in anhydrous pyridine/dichloromethane 50/50 (v/v)(20 mL). Cholesteryl chloroformate (Fluka, 3.0 g, 6.68 mmol) wasdissolved in anhydrous dichloromehthane (20 ml) and added slowly underargon with a syringe to the stirring reaction mixture. The mixture wasstirred under argon at room temperature for 2 h after which it wasconcentrated in vacuo. Residual DMF was coevaporated with toluene. Theresidue was dissolved in dichloromethane (50 mL) and washed with anequal volume saturated NaHCO₃. The aqueous layer was washed withdichloromethane and the combined organic extracts washed with an equalvolume saturated NaCl. The aqueous layer was washed with dichloromethaneand the combined organic layers dried over MgSO₄ and concentrated. Theresidue was chromatographed on a silica gel column with a gradient of25% ethyl acetate in hexanes to 100% ethyl acetate. The desired product(3.78 g, 60%) eluted with 100% ethyl acetate (R_(f) 0.41 by TLC)

EXAMPLE 18 Preparation of5′-O-(dimethoxytrityl)-2′-O-[hexyl-N-(3-oxy-carbonyl-cholesteryl)amino]uridine-3′-O-[2-cyanoethyl-N,N-di-isopropyl]Phosphoramidite

Nucleoside5′-O-(dimethoxytrityl)-2′-O-[hexyl-N-(3-oxycarbonyl-cholesteryl)amino]uridine(3.44 g, 3.3 mmol) was dissolved in dry dichloromethane (75 mL).2-cyanoethyl N,N,N′N′-tetraisopropylphosphorodiamidite (Sigma, 2.1 ml,6.6 mmol) and diisopropylamine tetrazolide (0.29 g, 1.7 mmol) were addedto the mixture, which was stirred under argon for 16H. Dichloromethane(75 mL) was added to the solution, which was washed with an equal volumeof saturated NaHCO₃. The aqueous layer was washed with an equal volumeof dichloromethane. The aqueous layer was washed with dichloromethane(30 ml) and the combined organic layers washed with an equal volume ofsaturated NaCl. The aqueous layer was washed with dichloro-methane (30mL) and the combined organic layers dried over Mg₂SO₄ and concentratedin vacuo. The residue was chromatographed on a silica gel column with agradient of 25% ethyl acetate in hexanes to 70% ethyl acetate. Thedesired product (3.35 g, 82% yield, R_(f)=0.71 by TLC in 50% ethylacetate in hexanes) eluted with 50% ethyl acetate.

EXAMPLE 19 Preparation of5′-O-(dimethoxytrityl)-2′-O-[hexyl-N-(3-oxycarbonyl-cholesteryl)amino]uridine-3′-O-(succinylaminopropyl)-controlled Pore Glass

Succinylated and capped controlled pore glass (0.3 grams) is added to2.5 ml anhydrous pyridine in a 15 ml pear-shaped flask. DEC (0.07 grams,0.36 mmol), TEA (100 μl, distilled over CaH₂), DMAP (0.002 grams, 0.016mmol) and5′-O-(dimethoxytrityl)-2′-O-[hexyl-N-(3-oxycarbonyl-cholesteryl)-amino]uridine(0.25 grams, 0.23 mmol) are added under argon and the mixture shakenmechanically for 16 hours. More nucleoside (0.20 grams) is added and themixture shaken an additional 18 hours. Pentachlorophenol (0.03 grams,0.11 mmol) is added and the mixture shaken 9 hours. CPG is filtered offand washed successively with dichloromethane, triethylamine, anddichloromethane. CPG is then dried under vacuum, suspended in 10 mlpiperidine and shaken 15 minutes. CPG is filtered off, washed thoroughlywith dichloromethane and again dried under vacuum. The extent of loadingis determined by spectrophotometric assay of dimethoxytrityl cation in0.3 M p-toluenesulfonic acid at 498 nm as approximately 39 μmol/g.

EXAMPLE 20 Preparation of5′-O-(dimethoxytrityl)-2′-O-[hexyl-N-(2,4-dinitrophenyl)amino] uridine

5′-O-(dimethoxytrityl)-2′-O-(hexylamino)uridine (0.88 grams, 1.37 mmol)was dissolved in methanol (20 mL). 2,4-Dinitrofluorobenzene (DNFB, 0.25grams, 1.37 mmol) was added and the mixture shaken on a mechanicalshaker. The reaction was monitored by TLC. After 90 min, another 0.25grams of DNFB was added and the reaction mixture shaken an additional 30min, followed by addition of another 0.25 grams of DNFB. After shaking2.5 hours, the mixture was concentrated in vacuo and chromatographed ona silica gel column, eluting with a gradient of 25% ethyl acetate inhexanes to 100% ethyl acetate. The desired product (0.51 grams, 46%)eluted with 100% ethyl acetate (R_(f) 0.85 by TLC).

EXAMPLE 21 Preparation of5′-O-(dimethoxytrityl)-2′-O-[hexyl-N-(2,4-dinitrophenyl)amino]uridine-3′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite

5′-O-(dimethoxytrityl)-2′-O-[hexyl-N-(2,4-dinitrophenyl)amino]uridine(0.45 grams, 0.55 mmol) was dissolved in dry dichloromethane (12 mL).2-Cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite (380 μL, 1.2mmol) and diisopropylamine tetrazolide (0.041 grams, 0. 024 mmol) wereadded to the mixture, which was stirred under argon for 16 hours. Thereaction mixture was then concentrated in vacuo and the residuedissolved in dichloromethane (75 mL) followed by washing with an equalvolume of saturated NaHCO₃. The aqueous layer was washed withdichloromethane (25 mL) and the combined organic layers washed with anequal volume of saturated NaCl. The aqueous layer was washed withdichloromethane (25 mL) and the combined organic layers dried over MgSO₄and concentrated. The residue was chromatographed on a silica gelcolumn, eluting with a gradient of 20% ethyl acetate in hexanes to 100%ethyl acetate. The desired product (510 mg foam, 93% yield, R_(f) 0.70by TLC) eluted with 100% ethyl acetate. ³¹PNMR (CDCl₃): 150.56 and150.82 ppm.

EXAMPLE 22 Preparation of5′-O-(dimethoxytrityl)-2′-O-[hexyl-N-(2,4-dinitrophenyl)amino]uridine-3′-O-(succinylaminopropyl) controlled Pore Glass

Succinylated and capped controlled pore glass (0.3 grams) is added to 3ml anhydrous pyridine in a 50 ml round-bottom flask. DEC (0.12 grams,mmol), TEA (25 μl, distilled over CaH₂), DMAP (0.005 grams, 0.041 mmol)and5′-O-(di-methoxytrityl)-2′-O-[hexyl-N-(2,4-dinitrophenyl)amino]uridine(0.21 grams, 0.26 mmol) are added under argon and the mixture shakenmechanically for 19 hours. More nucleoside (0.025 grams) is added andthe mixture shaken an additional 5.5 hours. Pentachlorophenol (0.045grams, 0.16 mmol) is added and the mixture shaken 18 hours. CPG isfiltered off and washed successively with dichloromethane,triethylamine, and dichloromethane. CPG then is dried under vacuum,suspended in 15 ml piperidine and shaken for 15 minutes. CPG is filteredoff, washed thoroughly with dichloromethane, and again dried undervacuum. The extent of loading is determined by spectrophotometric assayof dimethoxytrityl cation in 0.3 M p-toluenesulfonic acid at 498 nm, asapproximately 29 μmol/gm.

EXAMPLE 23 Preparation of5′-O-(dimethoxytrityl)-2′-O-[hexyl-N-(Nα-Nimid-Di-FMOC-L-Histidyl)amino]uridine

Nucleoside 5′-O-(dimethoxytrityl)-2′-O-(hexylamino)-uridine (0.97 g,1.51 mmol) was dissolved in dichloromethane (25 mL) and cooled to 0° C.in an ice bath. Nα,Nimid-Di-FMOC-L-histine pentafluorophenyl ester (2.4g, 3.1 mmol, purchased from Sigma) and 1-hydroxybenzotriazole (0.32 g,0.24 mmol, purchased from Fluka) were added to the stirred reactionmixture stirred under argon. After 15 minutes, the ice bath was removedand the mixture stirred under argon at room temperature for 72 h. Themixture was concentrated in vacuo and chromatographed on a silica gelcolumn, eluting with a gradient of 50% ethyl acetate in hexanes to 70ethyl acetate in hexanes. The desired product (0.53 g, 28%) eluted with70% ethyl acetate (R_(f) 0.53 by TLC in 100% ethyl acetate)

EXAMPLE 24A Preparation of5′-O-(dimethoxytrityl)-2′-O-[hexyl-N-(Nα-Nimd-Di-FMOC-L-histidyl)-amino]-uridine-3′-O-[2-cyanoethyl-N,N-diisopropyl]phosphoramidite

5′-O-Dimethoxytrityl-2′-O-[hexyl-N-(Nα-Nimid-Di-FMOC-L-histidyl)amino]uridine(1.9 g, 1.6 mmol) is dissolved in dry dichloromethane (20 mL).2-Cyanoethyl N,N,N′,N′-tetraiso-propylphosphorodiamidite (800 μL, 2.4mmol) and diisopropylamine tetrazolide (0.090 grams, 0.52 mmol) areadded to the mixture, which is stirred under argon for 20 hours. Thereaction mixture then is concentrated in vacuo and the residue dissolvedin dichloromethane (75 mL). The solution is washed with an equal volumeof saturated NaHCO₃. The aqueous layer is washed with dichloromethane(20 mL) and the combined organic layers washed with an equal volume ofsaturated NaCl. The aqueous layer is washed with dichloromethane (20 mL)and the combined organic layers dried over MgSO₄ and concentrated. Theresidue is chromatographed on a silica gel column, eluting with agradient of 50% ethyl acetate in hexanes to 100% ethyl acetate. Thedesired product elutes with 100% ethyl acetate.

EXAMPLE 24B Preparation of5′-O-(dimethoxytrityl)-2′-O-[hexyl-N-(Nα-Nimid-Di-FMOC)-L-histidyl)amino]uridine-3′-O-[succinylaminopropyl]controlled Pore Glass

Succinylated and capped controlled pore glass (dried under vacuum for 3hours immediately before use; 0.3 grams) is added to 3 ml anhydrouspyridine in a 50 ml round-bottom flask. DEC (0.12 grams, 0.63 mmol), TEA(25 μl, distilled over CaH₂), DMAP (0.005 grams, 0.04 mmol) and5′-O-(dimethoxy-trityl)-2′-O-[hexyl-N-(Nα-Nimid-Di-FMOC)-L-histidyl)amino]-uridine(0.21 grams, 0.17 mmol) are added under argon and the mixture shakenmechanically for 19 hours. More nucleoside (0.025 grams) is added andthe mixture shaken an additional 5.5 hours. Pentachlorophenol (0.045grams, 0.17 mmol) is added and the mixture shaken 18 hours. CPG isfiltered off and washed successively with dichloromethane,triethylamine, and dichloromethane. CPG then is dried under vacuum,suspended in 15 ml piperidine and shaken 15 minutes. CPG is filteredoff, washed thoroughly with dichloromethane and again dried undervacuum. The extent of loading is determined by spectrophotometric assayof dimethoxytrityl cation in 0.3 M p-toluenesulfonic acid at 498 nm. tobe approximately 27 μmol/g.

EXAMPLE 25 Preparation of5′-O-(dimethoxytrityl)-2′-O-[hexyl-N-(Ω-methyl-polyethyleneglycol-propionoyl)amino]uridine

Nucleoside 5′-O-(dimethoxytrityl)-2′-O-[hexylamino]-uridine, (1 g, 1.55mmol) is dissolved in anhydrous DMF (15 mL). 1-Hydroxybenzotriazole(0.24 g, 1.75 mmol) and polyethylene glycol-propionic acid-NHS-ester(1.23 g, 1.75 mmol) are added to the reaction mixture. The mixture isstirred under argon at room temperature for 2 hours after which it isconcentrated in vacuo. Residual DMF is coevaporated with toluene. Theresidue is dissolved in dichloromethane (50 mL) and then washed with anequal volume saturated NaHCO₃. The aqueous layer is washed withdichloromethane and the combined organic extracts washed with an equalvolume saturated NaCl. The aqueous layer is washed with dichloromethaneand the combined organic layers dried over MgSO₄ and concentrated. Theresidue is chromatographed on a silica gel column, eluting with agradient of 50% ethyl acetate in hexanes to 100% ethyl acetate. Thedesired product (1.08 g, 58% eluted with 100% ethyl acetate.

EXAMPLE 26 Preparation of5′-O-(dimethoxytrityl)-2′-O-[hexyl-N-{Ω-methyl-polyethyleneglycol-propionoyl)amino]uridine-3′-O-(2-cyano-ethoxy-N,N-diisopropyl}phosphoramidite

5′-O-(Dimethoxytrityl)-2′-O-[hexyl-N-(Q-methyl-polyethyleneglycol-propionoyl) amino] uridine (1.04 grams, 0.87 mmol) is dissolvedin dry dichloromethane (20 mL). 2-Cyano-ethylN,N,N′,N′-tetraisopropylphosphorodiamidite (800 μL, 2.4 mmol) anddiisopropylamine tetrazolide (0.090 grams, 0.52 mmol) are added to themixture, which is stirred under argon for 20 hours. The reaction mixturethen is concentrated in vacuo and the residue dissolved indichloromethane (75 mL). The solution is washed with an equal volume ofsaturated NaHCO₃. The aqueous layer is washed with dichloromethane (20mL) and the combined organic layers washed with an equal volume ofsaturated NaCl. The aqueous layer is washed with dichloromethane (20 mL)and the combined organic layers dried over MgSO₄ and concentrated. Theresidue is chromatographed on a silica gel column, eluting with agradient of 50% ethyl acetate in hexanes to 100% ethyl acetate. Thedesired product elutes with 100% ethyl acetate.

EXAMPLE 27 Preparation of5′-O-(dimethoxytrityl)-2′-O-[hexyl-N-(Ω-methyl-polyethyleneglycol-propionoyl) amino] uridine-3′-O-(succinyl-aminopropyl) controlledPore Glass

Succinylated and capped controlled pore glass (CPG) is dried undervacuum for 3 hours immediately before use. Controlled pore glass (0.3grams) is added to 3 ml anhydrous pyridine in a 50 ml round-bottomflask. DEC (0.12 grams, 0.67 mmol), TEA (25 μl, distilled over CaH2),DMAP (0.005 grams, mmol) and5′-O-(dimethoxytrityl)-2′-O-[hexyl-N-(w-methyl-polyethyleneglycol-propionoyl)amino]uridine (0.21 grams, 0.175 mmol) are added underargon and the mixture shaken mechanically for 19 hours. More nucleoside(0.025 grams) is added and the mixture shaken an additional 5.5 hours.Pentachlorophenol (0.045 grams, 0.17 mmol) is added and the mixtureshaken 18 hours. CPG is filtered off and washed successively withdichloromethane, triethylamine, and dichloromethane. CPG then is driedunder vacuum, suspended in 15 ml piperidine, and shaken 15 minutes. CPGis filtered off, washed thoroughly with dichloromethane, and again driedunder vacuum. The extent of loading is determined by spectrophotometricassay of dimethoxytrityl cation in 0.3 M p-toluenesulfonic acid at 498nm. to be approximately 18 μmol/g.

EXAMPLE 28 Preparation of Macrocycle Derivatized Nucleoside

5′-O-(dimethoxytrityl)-2′-O-(hexylamine)uridine is treated as per theprocedure of Example 3 with the macrocycle 4-{1,4,8,11-tetraza-[tri-(trifluoroacetyl)cyclotetradec-1-yl]}methyl benzoicacid-N-hydroxy succinimide ester (prepared according to Simon Jones, et.al., Bioconjugate Chem. 1991, 2, 416) to yield the product.

EXAMPLE 29 20 Preparation of Macrocycle Derivatized UridinePhosphoramidite

The nucleoside product of Example 28 is treated as per the procedure ofExample 4 to yield the product.

EXAMPLE 30 Preparation of Cpg Derivatized with Macrocycle DerivatizedNucleoside

The nucleoside product of Example 28 is treated as per the procedure ofExample 5 to yield the product.

EXAMPLE 31 Preparation of5′-O-(dimethoxyltrityl)-2′-O-(hexyl-N-(folate)-amino)uridine

5′-O-(Dimethoxytrityl)-2′-O-(hexylamine)uridine is treated as per theprocedure of Example 3 with folic acid pentafluorophenyl ester(protected with an isobutyryl protecting group) to yield the product.

EXAMPLE 32 Preparation of5′-O-(dimethoxyltrityl)-2′-O-[hexyl-N-(folate)-amino]uridine-3′-O-(2-cyanoethoxy-N,N-diisopropyl)phosphor-amidite

The nucleoside product of Example 28 is treated as per the procedure ofExample 4 to yield the product.

EXAMPLE 33 Preparation of CPG derivatized with5′-O-(dimethoxyltrityl)-2′-O-(hexyl-N-(folate)amino)uridine nucleoside

The nucleoside product of Example 31 is treated as per the procedure ofExample 5 to yield the product.

EXAMPLE 34 Preparation of5′-O-(dimethoxytrityl)-2′-O-{hexyl-N-[2-methoxy-6-chloro-9(Ω-amino-caproyl)acridine]amino}uridine.

6,9-Dichloro-2-methoxyacridine (Adlrich, 10 g, 36 mmol) and phenol (2.5g) were placed together on a round-bottom flask with a stirring bar andto this 6-amino-hexanoic acid (9.3 g, 71 mmol) was added and the flaskwas heated to 100° (oil bath) for 2 hours. TLC (10% methanol inmethylene chloride) showed complete disappearance of starting material.The reaction mixture was cooled and poured into 200 mL of methanol. Theproduct isolates out as a yellow solid (about 10 g). This compound wasthen converted into its pentafluorophenol ester.

5′-O-(Dimethoxytrityl)-2′-(hexylamino)uridine (0.5 g, 0.78 mmol) isdissolved in anhydrous DMF (15 mL). 1-Hydroxy-benzotriazole (0.16 grams,1.17 mmol) and 2-methoxy-6-chloro-9-(Ω-caproyl-amino) acridinepentafluorophenyl ester (0.53 grams, 1.17 mmol) are added to thereaction mixture. The mixture is stirred under argon at room temperaturefor 2 h, after which it is concentrated in vacuo. Residual DMF iscoevaporated with toluene. The residue is dissolved in dichloromethane(50 mL) and washed with an equal volume saturated NaHCO₃. The aqueouslayer is washed with dichloromethane and the combined organic extractswashed with an equal volume saturated NaCl. The aqueous layer is washedwith dichloromethane and the combined organic layers dried over MgSO₄and concentrated. The residue is chromatographed on a silica gel column,eluting with a gradient of 50% ethyl acetate in hexanes to 100% ethylacetate. The desired product elutes with 100% ethyl acetate.

EXAMPLE 35 Preparation of5′-O-(dimethoxytrityl)-2′-O-{hexyl-N-[2-ethoxy-6-chloro-9-(Ω-amino-caproyl)acridine]amino}uridine-3′-O-(2-cyanoethyl-N—N-diisopropyl)phosphoramidite

5′-O-Dimethoxytrityl-2′-O-{hexyl-N-[2-methoxy-6-chloro-9-(w-amino-caproyl)acridine]amino}uridine(0.80 grams, 0.77 mmol) is dissolved in dry dichloromethane (20 mL).2-Cyano-ethyl N,N,N′,N′-tetraisopropylphosphorodiamidite (800 μL, 2.4mmol) and diisopropylamine tetrazolide (0.090 grams, 0.52 mmol) areadded to the mixture, which is stirred under argon for 20 hours. Thereaction mixture is then concentrated in vacuo and the residue dissolvedin dichloromethane (75 mL). The solution is washed with an equal volumeof saturated NaHCO₃. The aqueous layer is washed with dichloromethane(20 mL) and the combined organic layers washed with an equal volume ofsaturated NaCl. The aqueous layer is washed with dichloromethane (20 mL)and the combined organic layers dried over MgSO₄ and concentrated. Theresidue is chromatographed on a silica gel column, eluting with agradient of 50% ethyl acetate in hexanes to 92% ethyl acetate. Thedesired product elutes with 100% ethyl acetate.

EXAMPLE 36 Preparation of5′-O-(dimethoxytrityl)-2′-O-{hexyl-N-[2-methoxy-6-chloro-9-(Ω-aminocaproyl)acridine]amino}uridine-3′-O-(succinyl aminopropyl) controlled Pore Glass

Succinylated and capped controlled pore glass (0.3 grams) is added to 3ml anhydrous pyridine in a 50 ml round-bottom flask. DEC (0.12 grams,0.67 mmol), TEA (25 μl, distilled over CaH₂), DMAP (0.005 grams, 0.04mmol) and5′-O-dimethoxytrityl-2′-O-{hexyl-N-[2-methoxy-6-chloro-9-(Ω-aminocaproyl)acridine]amino}uridine(0.21 grams, 0.17 mmol) are added under argon and the mixture shakenmechanically for hours. More nucleoside (0.025 grams) is added and themixture shaken an additional 5.5 hours. Pentachlorophenol (0.045 grams,0.17 mmol) is added and the mixture shaken 18 hours. CPG is filtered offand washed successively with dichloromethane, triethylamine, anddichloromethane. CPG is then dried under vacuum, suspended in 15 mlpiperidine and shaken 15 minutes. CPG is filtered off, washed thoroughlywith dichloromethane and again dried under vacuum. The extent of loadingis determined by spectrophotometric assay of dimethoxytrityl cation in0.3 M p-toluenesulfonic acid at 498 nm. to be approximately 27 μmol/g.

EXAMPLE 37 Preparation of5′-O-(dimethoxytrityl)-2′-O-[(hexyl-N,N-dimethyl)amino]uridine

5′-O-(dimethoxytrityl)-2′-O-(hexylamino)uridine (0.19 grams, 0.29 mmol)is dissolved in 4 ml methanol. Sodium acetate pH 4.0 (2 ml), sodiumcyanoborohydride (0.02 grams, 0.3 mmol) and 37% formaldehyde in water(300 μl) are added to the reaction mixture, which is stirred 2 hours,after which it is concentrated in vacuo. The residue is dissolved indichloromethane (50 mL) and washed with an equal volume saturatedNaHCO₃. The aqueous layer is washed with dichloromethane and thecombined organic extracts washed with an equal volume saturated NaCl.The aqueous layer is washed with dichloromethane and the combinedorganic layers dried over MgSO₄ and concentrated. The residue ischromatographed on a silica gel column, eluting with a gradient of 50%ethyl acetate in hexanes to 100% ethyl acetate. The desired product(0.15 grams, 80%) elutes with 10% Methanol-90% ethyl acetate.

EXAMPLE 38 Oligonucleotides Having 3′-Alkylamino Group

3′-O-Hexyl-(N-phthalimido)-aminouridine-CPG, i.e. the5′-O-dimethoxytrityl-3′-O-[hexyl-(Ω-N-phthalimidoamino)]-uridine-2′-O-(succinyl-aminopropyl) controlled pore glass fromExample 5, was used to synthesize the following oligonucleotides:

-   Oligomer 49: GAC U*-   Oligomer 50: GCC TTT CGC GAC CCA ACA CU-   Oligomer 51: GCC TTT CGC GAC CCA ACA CU*    wherein “*” denotes the 3′-O hexylamino-modified nucleoside.

Standard commercial phosphoramidites were used with the synthesis cycletimes specified by the manufacturer in a 380B ABI instrument (AppliedBiosystems).

Oligomer 49 was used for structural proof of 3′-O-alkylamine-bearingoligonucleotides at the 3′-terminal end. It showed the expected three³¹p NMR signals (−0.5 ppm, −0.25 ppm, −0,2 ppm) and seven lines in thetrace aromatic base region in ¹H NMR its spectrum.

Oligomer 51 was used to demonstrate the nuclease resistance offered bythis the alkylamino group and also for further conjugation. The oligomerwas treated with pyrene-butyric acid-N-hydroxy succinimide ester in 0.2M NaHCO₃ buffer/DMF. The product, Conjugate 1, was purified by HPLC andsize exclusion methods. HPLC retention times (eluting with a gradient of5% CH₃CN for 10 minutes then 5%-40% CH₃CN for 50 minutes) were asfollows:

Retention Time (min.) Oligomer 50 25.99 Oligomer 51 25.91 Conjugate 149.35

The nuclease stability of Oligomer 51 and the conjugate were testedagainst Oligomer 50 in HeLa cytoplasmic/nuclear extracts. The cellextract was diluted 1.4 times. The final concentration ofoligonucleotide was 20 μM. The half lives 10 of the oligonucleotideswere as follows:

t_(1/2) (hours) Oligomer 50 1.0 Oligomer 51 3.5 Conjugate 1 3.6

The half life of phosphodiester Oligomer 50 increased 3-4 times bysimple modification at the 3′-end with the hexylamino group, by itself,or by further conjugation.

EXAMPLE 39 Oligonucleotides Having 2′-O-Alkylamino Group

A. The phosphoramidite from Example4,5′-O-(dimethoxytrityl)-2′-O-[hexyl-(Ω-N-phthalimido)amino]-uridine-3′-O-[(2-cyanoethyl)-N,N-diisopropyl]phosphoramiditewas made as a 0.2 M solution in anhydrous CH₃CN and used to synthesizethe following oligonucleotides in an ABI DNA synthesizer, model 380 B.During the modified amidite coupling, the reaction time was increased to10 minutes. A coupling efficiency of approximately 90% was observed.After deprotection with concentrated ammonium hydroxide (55° C., 16hours) the oligonucleotides were purified by reverse phase HPLC anddesalting column (Sephadex G-25).

-   -   Oligomer 52. (SEQ ID NO:14): GCGTGU*CTGCG    -   Oligomer 53: GAU*CT

B. GCGTGTU′CTGCG where U′ is2′-O-[hexyl-N-(1-pyrene-propyl-carbonyl)amino uridine, Conjugate 2(Oligomer 52-pyrene butyrate conjugate).

To 20 O.D. of Oligomer 52 in 200 μL of 0.2 M NaHCO₃ buffer, 5 ml ofpyrene-butyric acid-N-hydroxy succinimide ester in an Eppendorf tube wasadded followed by 200 μL of DMF. The tube was shaken overnight. Thereaction was purified by size exclusion and HPLC to yield 18 O.D. ofproduct.

C. GCGTGTU″CTGCG where U″ is 2′-O-[6-bromoacetymido-hex-1yl]-uridine,Conjugate 3 (Oligomer 52-bromoacetate conjugate).

To 12 O.D. of Oligomer 52 in 100 μL of 0.2 M NaHCO₃ buffer, 2 mgbromoacetic acid-NHS ester (N-hydroxy succinimidyl bromoacetate) wasadded. After leaving the reaction to stand overnight, it was purified bysize exclusion and HPLC to yield 7.5 O.D. of product.

D. GCGTGTU^CTGCG where U^ is 2′-O-[hexyl-N-(polyethyleneglycol)-propionoyl]amino uridine, Conjugate 4 (Oligomer 52-PEGconjugate).

To 24 O.D. of Oligomer 52 in 200 AL of 0.2 M NaHCO₃ buffer, 20 mg ofPolyethylene glycol propionic acid-N-hydroxy succinimide ester wasadded. The reaction was mechanically shaken overnight and purified bySephadex G-25 size exclusion and chromatography to yield 22 O.D. ofproduct.

HPLC retention times (eluting with a gradient of 5% CH₃CN for 10 minutesthen 5%-40% CH₃CN for 50 minutes in a C-18 Delta-Pak reverse phasecolumn) were as follows:

Retention Time (min.) Oligomer 52 24.05 Conjugate 2 40.80 Conjugate 326.04 Conjugate 4 55.58

Changes in T_(m) due to pyrene conjugation were evaluated against bothDNA and RNA. T_(m) was measured in 100 mM Na⁺, 10 nM phosphate, 0.1 mMEDTA, pH 7 at 4 μM strand concentration.

The results were as follows:

T_(m) v. DNA (° C.) T_(m) v. RNA (° C.) Oligomer 52 50.9 55.5 Conjugate2 55.3 55.5 (4.4) (0.0)

The values in parentheses are changes in T_(m) compared to amino linkerin oligomer 52 as a control.

EXAMPLE 40 Oligonucleotide synthesis using2′-O-hexylamino(pyrene-butyrate)uridine phosphoramidite

The amidite 5′-O-(dimethoxytrityl)-2′-O-[hexyl-N-(1-pyrene propylcarbonyl)amino]uridine-3′-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (0.2M in anhydrous acetonitrile) was used to synthesizethe following oligomers, both for NMR studies:

-   Oligomer 54: GAU*CT-   Oligomer 55: GCC GU*G TCG-   (U*=2′-O-modified phosphoramidite)

These oligomers were purified trityl-on reverse-phase HPLC, detritylatedin 80% acetic acid for one hour and then repurified by RP-HPLC anddesalted by size-exclusion chromatography. NMR analysis showed thepresence of pyrene peaks.

EXAMPLE 41 Oligonucleotide synthesis using2′-O-hexylamino(dinitro-phenyl)uridine phosphoramidite

The amidite5′-O-(dimethoxytrityl)-2′-O-[hexyl-N-(2,4-dinitrophenyl)amino]uridine-3′-O-(2-cyanoethyl-N,N,-diisopropyl)phosphoramidite(0.18 M in anhydrous acetonitrile) was used to synthesizeoligonucleotides, Oligomers 56 to 63. All are analogues of an ICAMantisense sequence. These oligomers were purified trityl-on by RP-HPLC(Waters Delta-Pak C₁₈ column, 300 Å, 7.8 mm×30 cm, linear 50-mingradient of 5-60% acetonitrile in 0.05 M TEAA pH 7.3), detritylated in80% acetic acid for one hour and then purified by RP-HPLC and desaltedby size-exclusion chromatography. Data are summarized below:

Retention Back- Total Time bone (O.D.) (min) Oligomer 56: P = O 40 39.16GAU*CT Oligomer 57: P = S 64 39.19 (SEQ ID NO:15) U*GG GAG CCA TAG CGAGGC# Oligomer 58: P = S 45 39.21 U*GG GAG CCA TAG CGA GGC Oligomer 59: P= O 60 37.68 U*GG GAG CCA TAG CGA GGC Oligomer 60: P = O 69 38.58 U*GGGAG CCA U*AG CGA GGC (SEQ ID NO:5) Oligomer 61: P = O 86 32.38 TGG GAGCCA U*AG CGA GGC (SEQ ID NO:16) Oligomer 62: P = O 34 35.76 U*CT GAG TAG CAG AGG AGC TC# (SEQ ID NO:17) Oligomer 63: P = S 72 43.37U*GG GAG CCA U*AG CGA GGC# # = Non-nucleoside 6-carbon amino linker(Glen Research) and Bold indicates nucleotides having 2′-O-methylsubstitutions

EXAMPLE 42A Oligonucleotide synthesis using2′-O-[hexylamino-(cholesterol)]uridine phosphoramidite

The amidite5′-O-dimethoxytrityl-2′-O-[hexyl-N-(3-oxycarbonyl-cholesteryl)amino]uridine-3′-O-[2-cyanoethyl-N,N,-diisopropyl]-phosphoramidite(0.2M in anhydrous aceto-nitrile/dichloromethane 2:1 v/v) was used tosynthesize Oligomers 67-74. These oligomers are purified trityl-on byreverse-phase HPLC (Waters Delta-Pak C₁₈, 300 Å, 7.8 mm×30 cm, linear55-min gradient of 5-80% acetonitrile in 0.05 M TEAA pH 7.3),detritylated in 80% acetic acid for one hour and then repurified byRP-HPLC and desalted by size-exclusion chromatography. Data aresummarized below:

Reten- tion Back- Time bone Target (min) Oligomer 67: P = O NMR 52.73GAU*CT Oligomer 68: P = O ICAM 49.64 U*GG GAG CCA TAG CGA GGC Oligomer69: P = S ICAM 51.98 U*GC CCA AGC TGG CAT CCG TCA (SEQ ID NO:18)Oligomer 70: P = S CMV 52.57 U*GC GTT TGC TCT TCT TCT TGC G (SEQ IDNO:19) Oligomer 71: P = S mseICAM 53.24 U*GC ATC CCC CAG GCC ACC AT (SEQID NO:20) Oligomer 72: P = S Raf 53.95 U*CC CGC CTG TGA CAT GCA TT (SEQID NO:21) Oligomer 73: P = S PKCa 51.04 GU*T CTC GCT GGT GAG TTT CA (SEQID NO:22) Oligomer 74: P = S ICAM 52.75 F1-UU*GG GAG CCA TAG CGA GGC(SEQ ID NO:23) F1-U = U 2′-modified with fluorescein (see Example 42).

EXAMPLE 42B Synthesis of oligonucleotides using2′-O-[hexylamino-(fluorescein)] amidite

The amidite5′-O-dimethoxytrityl-2′-O-[hexyl-N-(5-thiocarbonyl-3,6-dipivolylfluorescein)amino]uridine-3′-O-(cyanoethyl-N,N-diisopropylphosphoramidite) (0.2 M in anhydrous acetonitrile) was used tosynthesize Oligomer 74 (above) and Oligomers 75-82 on a 1×10⁵ (Oligomer75) or 1×10² (remaining Oligomers) μmol scale. These oligomers arepurified trityl-on by reverse phase HPLC (Waters Delta-Pak C₁₈, 300 Å,7.8 mm×30 cm, linear gradient of acetonitrile in 0.05 M TEAA pH 7.3),detritylated in 80% acetic acid for one hour and then repurified byRP-HPLC and desalted by size-exclusion chromatography.

Backbone Target Oligomer 75: P = O NMR GAU*CT Oligomer 76: P = O ICAMUGG GAG CCA TAG CGA GGC Oligomer 77: P = S ICAM U*GC CCA AGC TGG CAT CCGTCA Oligomer 78: P = S ICAM U*GC CCA AGC TGG CAT CCG TCA# Oligomer 79: P= S CMV U*GC GTT TGC TCT TCT TCT TGC G Oligomer 80: P = S mseICAM U*GCATC CCC CAG GCC ACC AT Oligomer 81: P = S mseICAM U*GC ATC CCC CAG GCCACC A (U-CPG) (U-CPG) = 2′-O-hexylphthalimido U 6 Oligomer 82: P = S PKCGU*T CTC GCT GGT GAG TTT CA Where U* is U modified with fluorescein.

EXAMPLE 43 3-Benzyloxymethyl-3′-benzyloxymethyl-5′-O-tert-butyldiphenylsilylthymidine

To a mechanically stirred solution of5′-O-tert-butyldi-phenylsilylthymidine (170 g, 350 mmol) anddiisopropylethylamine (200 g, 1547 mmol) in methylene chloride (1000 ml)was added dropwise benzyl chloromethylether (171 g, 1092 mmol). Uponcompletion of a mild exotherm, the reaction was heated to 40° C. for 16h. Whereupon the reaction was washed with cold 5% HCl, H₂O, sat. NaHCO₃,dried (MgSO₄) and concentrated in vacuo. The resulting oil waschromatographed on silica gel (EtOAc/hexane, 8/2) to afford the productas a viscous oil, 251 g (71%). ¹H NMR(CDCl₃) 1.09 (s, 9H, (CH₃)₃), 1.60(s, 3H, C5—CH₃), 2.05 (ddd, 1H, C2′b), 2.52 (ddd, 1H, C2′a), 3.81 (dd,1H, C5′HH), 3.94 (dd, 1H, C5′HH), 4.08 (m, 1H, C4′H), 4.5 (m, 1H, C3′H),4.61 (s, 2H, OCH₂Ph), 4.72 (s, 2H, OCH₂Ph), 4.80 (s, 2H, OCH₂O), 5.51(s, 2H, NCH₂O), 6.39 (m, 1H, C1 H), 7.26-7.5 (m, 21H, CH═,ArH). Anal.Calcd. for C₄₂H₄₈N₂O₇Si: C, 69.97; H, 6.71; N, 3.89. Found: C, 69.81; H,6.42; N, 3.91.

EXAMPLE 44 3-Benzyloxymethyl-3′-benzyloxymethylthymidine

A solution of3-benzyloxymethyl-3′-benzyloxymethyl-5′-O-tert-butyldiphenylsilylthymidine(20 g, 28 mmol) in THF (200 10 ml) was treated with tert-butyl ammoniumfluoride 1M/THF (40 ml, 40 mmol) at room temperature for 16 hrs. Thesolution was concentrated in vacuo and the resulting oil chromatographedon silica gel (EtOAc/hexane, 7/3→8/2) to afford the product, 10 g (75%).m.p. 83-84° C.; ¹H NMR (CDCl₃), 1.92 (s, 3H, C5—CH₃), 2.20-2.50 (m, 3H,C2′H, C5′OH), 3.73 (dd, 1H, C5′HH), 3.89 (dd, 1H, C5′HH), 4.09 (m, 1H,C4′H), 4.49(m,1H, C3′H) 4.62 (s, 2H, OCH₂Ph), 4.70 (s, 2H, OCH₂Ph), 4.81(s, 2H, OCH₂O), 5.49 (s,2H, NCH₂O), 6.19 (t, 1H, Cl′H), 7.26-7.37 (m,5H, CH═,ArH). Anal. Calcd. for C₂₆H₃₀N₂O₇: C, 64.94; H, 6.26; N, 5.75.Found: C, 64.71; H, 6.27; N, 5.81.

EXAMPLE 45 3-Benzylocxymethyl-3′-benzyloxymethylthymidine-5′-aldehyde

A solution of 3-benzyloxymethyl-3′-benzyloxymethylthymidine (14.5 g, 30mmol) in DMSO (200 ml) was treated with DCC (33 g, 160 mmol) andphosphoric acid 85% (2.0 g) for 16h. The reaction mixture was filteredand concentrated in vacuo. The resultant oil was chromatographed onsilica gel (EtOAc/hexane, 7/3) to afford the product as a viscous oil,11 g (76%). ¹H NMR (CDCl₃) 1.92 (s, 3H, C5—CH₃), 2.20-2.52 (m, 2H,C2′H), 4.09 (m, 1H, C4′H), 4.49 (m, 1H, C3′H), 4.62 (s, 2H, OCH₂Ph),4.70 (s, 2H, OCH2Ph), 4.80 (s, 2H, OCH₂O), 5.50 (s, 2H, NCH₂O), 6.28 (t,1H, Cl′H) 7.24-7.51 (m, 11H, ArH, CH═), 9.65 (s, 1H, CHO). Anal. Calcd.for C₂₆H₂₈N₂O₇: C, 64.99; H, 5.87; N, 5.83. Found: C, 64.68; H, 5.95; N,6.01.

EXAMPLE 463-Benzyloxymethyl-3′-O-benzyloxymethyl-5′-deoxy-5′-N-(octa-decylamino)thymidine

A suspension of3-benzyloxymethyl-3′-benzyloxymethylthymidine-5′-aldehyde (11 g, 23mmol) and molecular sieve-4a (12 g) in tetrahydrofuran (250 ml) wastreated with octadecylamine (8 g, 30 mmol) for 16 hrs at roomtemperature. The mixture was then treated with sodium cyanoborohydride(95!k, 2.2 g, 33 mmol) and let stir an additional 16 hrs. The reactionmixture was filtered, concentrated in vacuo, partitioned betweenEtOAc/H₂O, separated, dried and reconcentrated in vacuo. The resultantgum was chromatographed on silica gel to afford a white powder.Recrystallization (MeOH) yielded the product, 3.8 g (23%). m.p. 60-62°C., ¹NMR (CDCl₃).88 (m, 3H, CH₃), 1.22-1.51 (m, 35H, CH₂), 1.93 (s, 3H,C5—CH₃), 2.07 (ddd, 1H, C2′a), 2.46 (ddd, 1H, C2′b), 2.51-2.94 (m, 4h,CH₂NH, C5′H), 4.07 (m, 1H, C4′H), 4.28 (m, 1H, C3′H), 4.62 (s, 2H,OCH₂Ph), 4.70 (9, 2H, OCH₂Ph), 4.80 (s, 2H, OCH₂O), 5.50 (s, 2H, NCH₂O),6.28 (t, 1H, Cl′H), 7.25-7.40 (m, 11H, CH═,ArH). Anal. Calcd. forC₁₄H₆₅N₃O₆: C, 72.19; H, 8.95; N, 5.74. Found: C, 71.88; H, 8.72; N,6.01.

EXAMPLE 473-Benzyloxymethyl-3′-O-benzyloxymethyl-5′-deoxy-5′-N-(octa-decylaminotrifluoroacetyl)thymidine

To a solution of3-benzyloxymethyl-3-O-benzyloxymethyl-5′-deoxy-5′-N-(octadecylamino)thymidine(5.8 g, 79 mmol) and TEA (4.0 ml, 28 mmol) in CH₂CH₂ (150 ml) was addedtrifluoroacetic anhydride (1.2 ml, 85 mmol). After 2h, TLC indicatedcompleteness of reaction. The reaction was concentrated in vacuo <40° C.and coevaporated with MeOH (2×25 ml). Chromatography on silica gel(EtOAc/hexane, 1/1) afforded the product, 6.4 g (98%). ¹H NMR (CDCl₃).88(m, 3H, CH₃), 1.25 (m, 32H, CH₂), 1.55 (m, 2H, CH₂CH₂NH), 1.93 (s, 3H,C5—CH₃), 2.10-2.51 (m, 4H, C2′H, CH₂NH), 3.22-3.82 (m, 2H, C5′H), 4.21(m, 2H, C3′H, C4′H), 4.63 (s, 2H, OCH₂Ph), 4.70 (s, 2H, OCH₂Ph), 4.80(s, 2H, OCH₂O), 5.50 (s, 2H, NCH₂O), 6.27 (t, 1H, C1′H), 7.23-7.41 (m,11H, ArH); ¹⁹F NMR (CDCl₃)-74.68, (DMSO-d₆)-69.36. Anal. Calcd. forC₄₆H₆₆F₃N₃O₇: C, 66.56; H, 8.01; N, 5.06. Found: C, 66.41; H, 7.74; N,5.29.

EXAMPLE 48 5′-Deoxy-5-N-(octadecylaminotrifluoroacetyl)thymidine

A suspension of3-benzyloxymethyl-3′-O-benzyloxymethyl-5′-deoxy-5-N-(octadecylaminotrifluoroacetyl)thymidine (5.5 g, 66 mmol) in methanol (250 ml), acetone (35 ml), aceticacid (0.5 ml) and palladium hydroxide/carbon (Pearlman's catalyst, 5.5g) was hydrogenated in a paar bottle for 48 hrs at 50 psi. The catalystwas filtered off on a celite bed and the celite washed carefully withhot acetone (4×200 ml). The filtrates were combined, concentrated invacuo to a solid and recrystallized (MeOH) to afford the product, 3.2 g(82%). m.p. 170-172° C. ¹H NMR (DMSO-d₆) 88 (m, 3H, CH₃), 1.23 (m, 32H,CH₂), 1.55 (m, 2H, CH₂CH₂NH), 1.80 (s, 3H, C5—CH₃), 2.07 (ddd, 1H,C2′a), 2.45 (ddd, 1H, C2′b), 3.30-3.87 (m, 6H, C2′H, CH₂CH₂NH, C5′CH₂),3.96 (m, 1H, C4′H), 4.15 (m, 1H, C3′H), 5.20 (m, 1H, C3′OH), 6.18 (t,1H, C1′H), (20° C.) 7.50 (s, 1H, CH═) and 7.55 (s, 1H,CH═), (90° C.)7.40 (s, 1H, CH═), 11.31 (s, 1H, ArNH), ¹⁹F NMR (DMSO)-69.2. Anal.Calcd. for C₃₀H₅₀N₃O₅F₃: C, 61.10; H, 8.54; N, 7.12. Found: C, 60.93; H,8.51; N, 7.34.

EXAMPLE 49 5′-deoxy-5′-N-(octadecylaminotrifluoroacetyl)thymidine-3′-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidite

A solution of 5′-deoxy-5′-N-(octadecylaminotrifluoro-acetyl)thymidine(5.9g, 10 mmol) in dry THF (1000 ml) was treated withbis-N,N-diisopropylaminocyanoethyl phosphite (8.0 g, mmol) andN,N-diisopropylaminotetrazolide (0.5 g, cat. amount) at rm. temp. for 16h. The reaction was concentrated in vacuo and the residue waschromatography on silica gel (hexane/EtOAc, 6/4) to afforded the productas a foam (5.1 g). ¹⁹F NMR (CDCl₃)−74.65; ³¹p NMR (CDCL₃) 149.63,149.56.

EXAMPLE 50

Plasma Uptake and Tissue Distribution of Oligonucleotides in Mice

The oligonucleotide, SEQ ID NO. NO:20 (oligomer 71), from example 42Awas used as a first test oligonucleotide. This olignucleotides isidentified in the figures as Isis 8005. Further oligonucleotide of thesame sequence were prepared in the same manner. These furtheroligonucleotides include a phosphorothioate oligonucleotide identifiedin the figures as Isis 3082 and an oligonucleotide incorporating a C₁₋₈alkyl group linked to the 5′ position of the nucleotides via a 5′ aminogroup (prepared utilizing the compound of Example 49 in the same manneras per the procedure of example 42) identified in the figures as Isis9047. The oligonucleotides were tritiated as per the procedure of Grahamet al., Nuc. Acids Res., 1993, 16, 3737-3743.

Animals and Experimental Procedure

For each oligonucleotide studied, twenty male Balb/c mice (CharlesRiver), weighing about 25 gm, were randomly assigned into one of fourtreatment groups. Following a one-week acclimation, mice received asingle tail vein injection of ³H-radiolabeled oligonucleotide(approximately 750 nmoles/kg; ranging from 124-170 μCi/kg) administeredin phosphate buffered saline, pH 7.0. The concentration ofoligonucleotide in the dosing solution was approximately 60 μM. Oneretro-orbital bleed (at either 0.25, 0.5, 2, or 4 hours post-dose) and aterminal bleed (either 1, 3, 8 or 24 hours post-dose) was collected fromeach group. The terminal bleed was collected by cardiac puncturefollowing ketamine/xylazine anesthesia. An aliquot of each blood samplewas reserved for radioactivity determination and the remaining blood wastransferred to an EDTA-coated collection tube and centrifuged to obtainplasma. Urine and feces were collected at intervals (0-4,4-8 and 8-24hours) from the group terminated at 24 hours.

At termination, the liver, kidneys, spleen, lungs, heart, brain, sampleof skeletal muscle, portion of the small intestine, sample of skin,pancreas, bone (both femurs containing marrow) and two lymph nodes werecollected from each mouse and weighed. Feces were weighed, thenhomogenized 1:1 with distilled water using a Brinkmann Polytronhomogenizer (Westbury, NY). Plasma, tissues, urine and feces homogenatewere divided for the analysis of radioactivity by combustion and fordetermination of intact oligonucleotide content. All samples wereimmediately frozen on dry ice after collection and stored at −80° C.until analysis.

Analysis of Radioactivity in Plasma, Tissue, and Excreta

Plasma and urine samples were weighed directly into scintillation vialsand analyzed directly by liquid scintillation counting after theaddition of 15 ml of BetaBlend (ICN Biomedicals, Costa Mesa, CA). Allother samples (tissues, blood and homogenized feces) were weighed intocombustion boats and oxidized in a Biological Materials Oxidizer (ModelOX-100; R. J. Harvey Instrument Corp., Hillsdale, NJ). The ³H₂o wascollected in 20 ml of cocktail, composed of 15 ml of BetaBlend and 5 mlof Harvey Tritium Cocktail (R. J. Harvey Instrument Corp., Hillsdale,NJ). The combustion efficiency was determined daily by combustion ofsamples spiked with a solution of ³H-mannitol and ranged between73.9-88.3%. Liquid scintillation counting was performed using a BeckmanLS 9800 or LS 6500 Liquid Scintillation System (Beckman Instruments,Fullerton, Calif.). Samples were counted for 10 minutes with automaticquench correction. Disintergration per minute values were corrected forthe efficiency of the combustion process.

Analysis of Data

Radioactivity in samples was expressed as disintergrations per minuteper gram of sample. These values were divided by the specific activityof the radiolabel to express the data in nanomole-equivalents of totaloligonucleotide per gram of sample, then converted to percent of doseadministered per organ or tissue. Assuming a tissue density of 1 gm/ml,the nmole/gram data were converted to a total μM concentration. Tocalculate the concentration of intact oligonucleotide in plasma, liveror kidney at each time point, the mean total μM concentrations weredivided by the percent of intact oligonucleotide in the dosing solution(82-97%), then multiplied by the mean percentage of intactoligonucleotide at each time point as determined by CGE or HPLC. Thesedata was then used for the calculation of tissue half-lives by linearregression and to compare the plasma pharmacokinetics of the differentmodified oligonucleotides. The pharmacokinetic parameters weredetermined using PCNONLIN 4.0 (Statistical Consultants, Inc., Apex, NC).After examination of the data, a one-compartment bolus input, firstorder output model (library model 1) was selected for use.

The result of the animal plasma uptake and tissue distribution tests areillustrated graphically in FIGS. 1, 2, 3 and 4. As is seen in FIG. 1,plasma concentration of each of the test oligonucleotides decrease fromthe initial injection levels to lower levels over the twenty-four hourtest period. Plasma concentrations of the two oligonucleotides bearingconjugate groups of the invention were maintained at a higher level fora longer period than were those of the non-conjugate bearingphosphorothioate All of the test compounds were taken up from the plasmato tissues as is shown in FIGS. 2, 3 and 4. The two compounds of theinvention had different distribution between the various tissues. FIG. 2shows the tissue distribution of the standard oligonucleotide, ISIS3082. FIG. 3 shows the tissue distribution of oligonucleotides ISIS 9047while FIG. 4 shows the tissue distribution of oligonucleotide ISIS 8005.

1. A compound which hybridizes with a nucleic acid and comprises aplurality of linked nucleosides, wherein: each nucleoside includes apentofuranosyl sugar portion and a base portion; and at least one ofsaid nucleosides bears at a 2′-O-position, a 3′-O-position, or a5′-O-position of said pentofuranosyl sugar, a terminal substituenthaving formula:—R_(A)—N(R_(1a))(R_(1b)) where: R_(A) is alkyl having from 1 to about 10carbon atoms or (CH₂—CH₂—Q)_(x); R_(1a) and R_(1b), independently, areH, R₂, or an amine protecting group or have formula C(X)—R₂,C(X)—R_(A)—R₂, C(X)—Q—R_(A)—R₂, C(X)—Q—R₂; and R₂ is a peptide; X is Oor S; each Q is, independently, is NH, O, or S; and x is 1 to about 200.2. The compound of claim 1, wherein more than one of said nucleosidesbear said substitution at a 2′-O-position, 3′-O-position, or a5′-O-position.
 3. The compound of claim 1, wherein R_(A) is (CH₂)_(n)where n is an integer from 1 to about
 10. 4. The compound of claim 3,wherein n is
 6. 5. The compound of claim 1, wherein R_(1a) is H andR_(1b) is R₂.
 6. The compound of claim 1, wherein said peptide comprisesone or more amino acids.